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PHYSIOLOGICAL
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THE AMERICAN
VOLUME 35
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PHYSIOLOGICAL
JANUARY
Mechanism
REVIEWS
SOCIETY,
INC.
NUMBER
1955
I
of Action and Properties of Pyridine
Nucleotide-Linked
Enzymes’* *
E. RACKER
From
the Department
of Biochemistry,
New Haven,
Yale University
Connecticut3
Sclzool of Medicivte
HE substrates available as food for unicellular and multicellular organisms
are rather stable. Their utilization by the cell is therefore preceded by a
processof activation and degradation which is accomplishedby specific enzymes. The principal activities of these enzymes consist of oxidation-reduction,
cleavage, group transfer and removal or addition of water, phosphate, CO2 and
ammonia.
GENERALCONSIDERATIONS
Oxidation Reductions and Formation of High Energy Bonds
In the courseof these processesthe food substancesare transformed into compounds with high energy bonds (I) which can contribute to the pool of pyrophosphate nucleotides. In theselatter compoundsenergy is stored in a form immediately
available for the purpose of biosynthetic or mechanical work. Oxidative processes
contribute the major share of these energy rich bonds. At present at least six apparently different reaction types are known which lead to the formation of compounds with a high energy bond A F of hydrolysis of about - 8000caloriesor higher).
These reactions are listed in table I. It can be seenthat in the first four reaction
types a thiol ester is the primary product; this can then be transformed into an
acyl phosphate. In reaction 5 an enol phosphate is formed by the dehydration of a
phosfihorylated hydroxy-acid. In reaction 6, ATP* is formed by a seriesof reactions
called oxidative or coupled phosphorylations. The detailed mechanism of these latl Investigations carried out in the author’s laboratory and referred to in this review were
supported by grants from the U.S. Public Health Service and the National Research Council.
2 ABBREVIATIONS:
ADH, alcohol dehydrogenase; Ale, alcohol; Ald, aldehyde; AMP, adenosine$-phosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; BAL, 2,3-dimercapto
propanol; CoA, coenzyme A; DHA, dihydroxyacetone phosphate; DPN, diphosphopyridine nucleotide, coenzyme I; DPN-ase, diphosphopyridine nucleotidase; DPND, reduced deutero-DPN;
DPNH, reduced DPN; EDTA, ethylenediamine tetra-acetate; FAD, flavin-adenine dinucleotide;
G-6-p glucose-6-phosphate; GDP, guanosine diphosphate; GSH, glutathione; GTP, guanosine
triphosphate; IAA, iodoacetate; IDP, inosine diphosphate; ITP, inosine triphosphate; LDH, lactic
dehydrogenase; OAA, oxaloacetate; OSA, oxalosuccinate; PCMB, para-chloromercuribenzoate;
PEP, phosphoenol pyruvate; 6-Pg, 6-phosphogluconate; ThPP, thiamine pyrophosphate.
a Present address: The Public Health Research Institute of the City of New York, Inc., Foot of
East 15th Street, New York 9, N. Y.
E. RACKER
2
TABLE
Reaction
Type
of an aldehyde
to a thiol
I) Oxidation
&c=()
( +
R1C+SR2
a) Oxidation
of a ketoacid
:
Examples
Triose-p-dehydrogenase;
dehyde + acylenzyme)
‘Cofactors’
DP?\J’
and
GSH
(acetal-
CH3
1
e
H + C=(-j
R’C
1
SR2
CH3
I
:
ester
I
to a thiol
ester
Pyruvic
dehydrogenase
+ acetyl lipoic?
(pyruvic
Lipoic
acid
CO2 CH3
CH3
HOG&C=0
+
I
H
s
C--O
I
s
---)
&
R
3) Thioclastic
Thiolase
(acetoacetyl
acetyl CoA)
cleavage
SR
SR
I
C=O
H&-
CH3
I i
I
C=O
4) Oxidation-reduction
e
I
C=O
+ CHz
I
I
CH3
C=O
of ketoaldehyde
H
0
CH3-CyC=O
H
0
+ CHo-C-C=0
H
:
H+SR
i*
5) Dehydration
acid
CHzOH
of phosphorylated
CHOPO3Hz
COOH
6) Phosphorylation
tion
Glyoxalase
(Methyl
lactoylglutathione)
.glyoxal
GSH
I
SR
hydroxy-
Enolase
(z-phosphoglyceric
phosphoenolpyruvic)
CHz
C-OP03H2
COOH
coupled
to DPXH
oxida-
ter reactions is unknown, but the first successful attempts at a physical rractronation (2) and a functional separation of individual steps of the electron transportlinked phosphorylations
have been reported (3).
In reactions 1-4 which lead to formation of thiol esters the awld acceptors are
either glutathione, coenzyme A or lipoic acid. With a given enzyme the reactions
are usually restricted to only one of the mercaptans. Other SH compounds do not
substitute readily. Pyridine nucleotides are linked directly only to reactions I and
6, but participate in secondary reactions with the products of reacfions
z-5. In
1
I-
.
l
January
1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
3
the first four reactions the substrate donates its ‘acyl moiety’ to the sulfur of the
cofactor while the residual portion of the substrate combines with the portion of
the cofactor originally linked to the sulfur. The formation of citric acid from acetyl
CoA and oxaloacet,ate may be visualized as belonging to reaction type 3.
!
i COOH
HOiOC--CH*+C---CH2COOH
I1
i0
I
H--GXoA
/H
II
I
1
$
0
II
H:!O + CH3-- C-S-CoA
+ COOH COCH2COOH
The thioclastic cleavage of citric acid leading to the formation of acetyl CoA (cf. 4)
has the unique feature of condensinga carboxyl with an SH group resulting in the
formation of a high energy bond by dehydration.
All the reactions leading to the formation of high energy bonds may be considered as oxidation-reduction processesin a broad sense.The oxidation-reduction
may be an intramolecular reaction either in the substrate (e.g. 2-phosphoglycerate*
phosphoenolpyruvate) or by participation of the cofactor (e.g. S-S lipoic acid +
S-acyl-lipoic-SH). It may be an intermolecular reaction requiring the aid of a hydrogen carrier (e.g. DPN in triose phosphate oxidation). In the actual processwhich
results in the formation of the high energy bond this hydrogen carrier may act
either as a hydrogen acceptor (e.g. in triose phosphate oxidation) or as a hydrogen
donor (e.g. in coupled phosphorylation).
Since it is apparent that the oxidative step is the key reaction in the formation
of high energy bonds, the fate and the mode of transport of the electrons must be
known before the energy producing machinery of living cells can be understood.
Animal cells have a ‘hierarchy’ of electron carrier systems which are ranked according to their potentials from the strongly reducing lipoic acid to the high potential
of the cytochrome oxidase system. If the oxidation-reduction potential of the substrates (e.g. a ketoglutarate-succinate) is sufficiently low, the entire chain of carriers from lipoic acid to pyridine nucleotides to flavoprotein and to the cytochromes
may be employed. Frequently the chain of carriers is shorter either becauseof the
higher potential of the substrates (e.g. succinate-fumarate) or becauseof a short
circuit which serves to simplify the processat the expense of energy yield. Such
short circuits are probably used by the cells for specific synthesis or for the accumulation of a reductant (e.g. reduced glutathione or coenzyme A).
In some microorganismsthe complex electron transport system, characteristic
of animal cells, appears to be lacking. Instead, the hydrogen is transported by a
hydrogen carrier, usually a pyridine nucleotide, to an acceptor available in large
quantities (e.g. pyruvate, acetaldehyde, amino acids, S-S compounds, nitrates,
etc.). Frequently the reduced acceptors are excreted by the microorganisms as
waste products. High energy bonds may be formed by these anaerobic oxidationreductions, as in the case in the formation of I ,3-diphosphoglyceric acid during
glycolysis. The hydrogen acceptor must not only be available in amounts stoichiometric to the oxidized substrate, but should also have a higher oxidation-reduction
potential. The transfer of the hydrogen from the reduced carrier to the acceptor has
not been recorded in the literature to give rise to high energy bonds. However, such
4
E. RACKER
Volume 35
a possibility should be consideredand could be justified on theoretical grounds. It
may be suggested that an anaerobic coupled phosphorylation at the electron
transport level participates in the formation of high energy bonds in muscle. Such
a reaction may account for the unexpectedly high yield of up to 1.9 molesof creatine
phosphate per mole of lactic acid formed in living muscle under anaerobic conditions (5).
Chemistry of Pyridine Nucleotidesand SH-Cofactors
Comprehensive reviews are available on the chemistry and biosynthesis of
pyridine nucleotides (6-8) as well as on the chemistry of coenzyme A, glutathione
and lipoic acid (9-13).
Therefore only a few recent developments in this field and
somesingular chemical features, which have a bearing on the discussionof mode of
action of these cofactors, have been selected for a more detailed discussion. L4n
evaluation of the data on coenzyme III will not be presented, since the subject has
been reviewed elsewhere (8). This cofactor, obtained from yeast, appears to be
closely related to DPN and can be converted into DPN by absorption and elution
from charcoal (14). In view of this fact and the high reactivity of DPN with various
compounds,which will be discussedbelow, the possibility that coenzyme III is DPX,
reversibly complexed to another compound, may be considered.
Position of Hydrogen in Reduced DPN. Karrer and his collaborators have
studied the products of chemical reduction of N1-methylnicotinamide and the Wsubstituted pyridine derivatives (IS, 16). Reduction with sodium hydrosulfite and
comparison of the product with known ortho and para dihydro compoundsled them
to conclude that reduction of the pyridinium ring of DPN and of simpler N1-substituted nicotinamide derivatives occurred in the position ortho to the pyridinium
nitroge:n. Recent experiments demonstrate that the para position is the actual site
of reduction in the case of DPN (17) as well as in N1-methylnicotinamide (x8).
Fisher et al. (19) have observed that reduced DPN prepared with sodium hyhrosulfite in the presenceof heavy water transferred only about one-half of its deuterium
to acetaldehyde in the presence of yeast alcohol dehydrogenase. The reaction is
stereospecificsince the enzyme is capable of removing the hydrogen only from one
side of the plane of the pyridine ring. It was therefore possibleto obtain oxidized
DPN containing deuterium and to localize the position of the deuterium by the
following procedure (17, 20) : DPN was reduced by sodium hydrosulfite in the
presenceof heavy water and the isolated reduced DPN oxidized wit,h acetaldehyde
in the presenceof alcohol dehydrogenase.DPN was then cleaved by DPNase and
the nicotinamide isolated. After methylation with methyl iodide and oxidation with
alkaline ferricyanide the 2- and 6-pyridones were isolated and analyzed for deuterium.
The data obtained by this procedure ruled out reduction of DPN in. either
of the two ortho positions and strongly pointed to the position para to the nit,rogen
as the site of enzymatic reduction. Confirmation of this view has come from experiments of Vennesland, Westheimer and their collaborators (2 I). Xicotinamide labeled
with deuterium in the 2,~ or 6 position was incorporated into DPN by means of
the DPBiase reaction. After chemical reduction DPNH containing deuterium in
position 4 gave rise by enzymatic transfer with lactic dehydrogenaskto deuteriumlabeled lactate, while deuterium in the other positions did not.
The stereospecificutilization of hydrogen from one side of the pyridine ring as
shown in FomuZa A and B has been demonstrated for alcohol and lactic dehydro-
genase (Cf. 22). Recent
stereospecific utilization
t apparen
side of the nice #tinamide
experiments in Vennesland’s laboratory demonstrate the
of hydrogen in the case of bacterial testosterone dehydrotly with the bacterial enzyme the hyd .rogen from the &her
ring is transferred.
CONHZ
D\ 1
-;<-->W--R
CONH,
E-I I
>(-.-)X--R
(A>
(W
Adler et al. (24) reported
that addition of hydrosulfite to a DPN solution in 0.1 N KaOH under anaerobic
conditions gives rise to a yellow compound with an absorption peak around 360
mp. The a,uthorssuggestedthat this compound is either a one-stepreduction product
of DPX (e.g. a semiquinone radical) or an addition product of hydrosulfite and
DPN. It was not possible to reduce the compound with amalgams to DPXH as
might have been expected if a one-step reduction product had been formed, nor
was it possibleto detect a free radical by microwave spectroscopy (25). Yarmolinsky
and (Xowick (26) recently presented strong evidence for a third hypothesis. Since
they obtained IOO per cent transformation of the yellow compound into reduced
DPX under anaerobic conditions, they could rule out the one-stepreduction product,
which could maximally yield 50 per cent DPNH by an anaerobic oxidation-reduction process.With DzO, DPX and hydrosulfite it was shown that deuterium was
not incorporated into the yellow intermediate although it was present in DPXH,
the final -product of the reaction. The yellow compound, af ter- removal of excess
hvdrosuliite
and sulfite, was found to contain sulfur and on hydrolysis at neutral
a
pi1 yielded stoichiometric amounts of DPNH and sulfite.
In view of thesefindings and someother experimental observations with formaldehyde sulfoxalate, the authors suggest that hydrosulfite is first hydrolyzed to
sulfoxvlic- which reacts with DPN to form a sulfoxylate (DP&--O--S-O
Ka).
This ~Tellow
intermediate is then hydrolyzed to yield DPNH and sulfite as outlined
.
be1ow:
Mechanism
of Reduction
of DPN
H
by Hydrosulfite.
OSOH
+
H
H+
--+
Hz0
H
\/
/\/
,
COKH2
I’
I! ii
+
SOT +
2H+
The similarity of t.his reaction mechanismto that proposed for the interaction
of triose phosphate dehydrogenaseand DPN will be discussedlater.
6
?‘ollr?tle -75
E. RACKER
Chemical Interactions
With Pyridine Nucleotides and With SW Cofactors.
Meyerhof el aZ. (27) observed changes in the absorption spectrum of DPN on addition of cyanide or bisulfite. Interactions between acetone and alkali with N’-substituted nicotinamide derivatives were described by Najjar et ctL. (28);
Needham
et al. (29) reported a reaction between free trioses and DPN. These interactions were
studied in great detail by Colowick, Kaplan and their collaborators (30-32).
They
found that the interaction of DPN with cyanide leads to the appearance of an absorption band at 325 rnp and to a decrease of absorption at 260 rnp. The reaction
is readily reversed on dilution. DPN treated with 5 E; alkali gives rise to a compound with a high absorption at 340 rnp, followed by a secondary reaction to form
a fluorescent compound with a peak at 360 rnp (31). The reaction of DPN and dihydroxyacetone and hydroxyl ions gives rise to a compound with an absorption
spectrum quite similar to that of DPNH. The E340 = 6.3 X 10~ cm.2/mole is identical to that of DPNH (32). The compound exhibits an acid lability similar to that of
DPNH and it has the same RF in paper chromatograms. The non-identity with
DPNH was establishedby the lack of reaction with alcohol dehydrogenasein the
presenceof acetaldehyde. Dihydroxyacetone reacts only with pyridine compounds
which contain a quaternary nitrogen and an amide group. This fact and several
other features led the authors to suggestthat the reaction may serve as a model for
biological interactions with DPN. A detailed hypothesis for an interaction of TIIW
with phosphate in coupled phosphorylation was presentedseveral years ago (33).
Several agents have been shown to oxidize reduced DPN. o-Quinones (34),
phenazine dyes (35), riboflavins (36)) ferricyanide (37), m-dinit robenzene (38) and
2,Gdichlorophenolindophenol
(39) have been reported to react in the absenceof
enzymes. It should be pointed out however that some of these nonenzyrnatic reactions are quite sluggishand occur at appreciable rates only at. high concentrations
of the oxidant. At low concentrations, ferricyanide, 2,6-dichlorophenolindophenol
and several other dyes do not oxidize DPNH readily (40, 41). When quantitative
measurementsof dehydrogenase activities are carried out, particularly in purified
preparations, diaphorase must be added to insure that the oxidation of DPNH is
not the limiting step.
Reactions of SH compounds with various reagents have been ext-ensively reviewed by Barron (42). Sulfhydryl reactive compounds have been widely used in
studies of physiological mechanisms,e.g. the effect of iodoacetate on muscular contraction (43), or the effect of p-chloromercuribenzoate on bleaching and synthesis
of rhodopsin (44). ‘Sulfhydryl inhibitors’ have been very useful in the elucidation of
the mechanismsof enzyme action, e.g. of triose phosphate dehydrogenase (45) or
of thiolase (46). They have also been used in studies of t,he interaction of proteins
with pyridine and flavine nucleotides (47-51).
Three SH inhibitors,
narnel~
iodoTABLE
2. EFFECT
OF INHIBITORS
ON FREE
Iodoacetate,
TO Inhibition
GSH” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzyme
(charcoal treated) t . . . . . . . . . . . . . . .
DPN-enzymet
. . . . .. .... ... .... ... ... ...
AND
BOUND
GSH
Todoacetamide,
yc Inhibition
X-ethylmaleimide,
sj Inhibition
4
12
IO0
5
30
07
94
* Reaction
measured by nitroprusside
test. GSH: 0.067 p moles/ml.
t Reaction
measured by activity
tests. Enzyme:
0.03 p moles/ml.
Inhibitors:
incubated
for 12 min. at room temperature
in 0.01 M potassium
phosphate
PH 6.5.
26
o. T I ,U moles/ml.
Ja.nuary I 955
WRIDINE
NUCLEOTIDE-LINKED
ENZYMES
7
acetate, iodoacetamide and N-ethyl maleimide, have been recently compared in
regard t,o their reactivity with free and protein bound glutathione (so). It was found
that GSH and the SH group of triose phosphate dehydrogenase reacted at comparable rates with each of these inhibitors. Most reactive is N-ethyl maleimide, least
reactive is iodoacetate. But this order of reactivity was only found with enzyme
free of DPN. In the presenceof DPN the enzyme becamevery susceptibleto iodoacetate and lesssusceptibleto the maleimide (seetable 2). Thus it is clear that the
reactivity of SH inhibitors with proteins may largely be determined by properties
of adjacent groups.
Oxidation-Reduction
Potentials.
The oxidation-reduction potential of the DPN
and TPN system has been generally assumedto be about -0.28
volts. Accurate
measurementsand calculations indicate a more negative value of El (25O, PH 7.0)
c -0.32 volts for DPh’ and -0.324 for TPN (52). It is important to stressthat
the more recent data were obtained with crystalline enzymes, sincein crude systems
erroneousvalues due to sidereactions may be obtained. Alcohol dehydrogenasefrom
yeast, ca.talyzing the reactions ethanol C acetaldehyde and isopropanol + acetone,
and glutamic dehydrogenase from liver, catalyzing the reaction glutamic acid *
a ketoglutaric acid + NH3, were used in these studies. Calculations of Ei for some
biological dehydrogenations are listed in table 3 (52). Recent potentiometric measurements (53) in the presenceof milk xanthine oxidaseand benzylviologen as electromotively active mediator gave a value of -0.318 volts for the DPN system.
Potentiometric measurementsof SH-SS systems have not met general acceptance (cf. II). At,tempts to demonstrate the reversibility of the nucleotide-linked
reduction of oxidized glutathione have failed (54, 55). No reduction of TPN by reductase and GSH could be detected even when the reaction was coupled to a TPNH
oxidizing system such as provided by pyruvate in the presenceof lactic dehydrogenase (40). Such measurementsdemonstrate that the potential of the GSH system
must,be considerably higher than that of the DPN system. In view of the above, and
the reactivity of GSH with the ascorbic-dehydroascorbicsystem (cf. 56, 57), the potentiometric value of +0.03 volts (II)
for GSH-GSSG appears reasonable. On
the other hand data obtained with the DPN-linked lipoic acid dehydrogenase indicate a very low potential (-0.42
v.) for the lipoic acid system (13). This low potentia,l has been ascribed to the presenceof intramolecular SH groups in reduced
lipoic acid (I I). It is therefore apparent that SH compounds, depending on their
structure and the position of the SH groups, may cover a wide range of Eh values.
In view of the presenceof SH group in proteins capableof reducing DPN, a phenomenon to be discussedlater, a systematic analysis of the steric factors which govern
t,he potethls of SH--SS systems would be of considerable value for the underst,anding of the mode of action of some dehydrogenases.It is hoped that further
discoveries of nucleotide-linked enzymes which catalyze the oxidation of SH and
reduction of S-S compounds may permit equilibrium studies, thus supplementing
available potentiometric data on SH compounds.
Functional
Role
of” Thkol
and Pyridine
Nucleotide
Cofactors
The major role of sulfur containing cofactors appears to reside in their function as acyl group carriers, while the pyridine nucleotides have been looked upon as
hydrogen carriers. A rigid division of labor doesnot seemto exist however, as is apparent from the participation of lipoic acid and GSH in hydrogen transfer reactions.
Less widely known is the participation of pyridine nucleotide in reactions other
TABLE
3. Ei
Reductant
Isocitrate3+ H+
DPNH
+ H+
TPNH
-+- H+
Tsopropanol
Glyceraldehyde-3-phosphate”Ethanol
a-Glycerophosphate2L-Lactatex,-Malate
VALUES
FOR
+ HPOJ+
SOME
BIOLOGICAL
DEHYDROGENATWNS
Oxidant
Q! Retoglutarate*
DPN+
TPN+
Acetone
I, 3 Diphosphoglyceric4Acetaldehyde
Dihydroxyacetone
phosphat&
PyruvateOxaloacetate2-
(,sz)
---O.&
.%- o.j20
0.324
...- 0 * q6
-’ - 0.
286
--0.204
-.-0.192
--O.IC)O
- -0.
Ii,6
than oxidation-reductions.
A possible role of DPN in phosphorylation reactions was
suggested many years ago (58). The observation was made that DPN can substitute
for adenylic acid in the transphosphorylation
from phosphopyruvate
to glucose.
These findings have been dismissed on grounds of contamination with adenylic
acid either in the DPN preparation itself or due to enzymatic cleavage of DPN.
However, a pronounced inhibition of phosphorylation by Naf, which ockrred only
in the DPN-linked
system, appears to contradict the simple contamlination theory.
Recently it was found that DPN is required for phosphorolysis,
arsenolysis and
acyl group transfer catalyzed by triose phosphate dehydrogenase (45) as well as for
acetyl phosphate hydrolysis (59) which all occur in the absence of oxidation-reduction. In phosphorylation
coupled to DPNH oxidation an interaction between the
phosphate and the pyridine nucleotide appears likely. A reaction of DPN with
phosphate, analogous to that with cyanide, alkali or hydrosulfite, has been proposed
to play a part in coupled phosphorylation (33). In this connection it may be significant that DPN is firmly attached to some dehydrogenases as well as to mitochondria
and tissue particles (60) which carry out coupled phosphorylations.
To these observations bearing on secondary functions of nucleotide coenzymes
one may add the following findings: Adenosine-5’-phosphate
and adenosine-2’phosphate act as cofactors in dehydrogenase and transhydrogenase
reactions (61 t
62);
decarboxylation of oxaloacetate by ‘malic’ enzyme is either st.imulated or inhibited. by TPN depending on the source of enzyme (63, 64). 1’I’P st.imulate=; OAA
carboxylase (65). Adenosine-$-phosphate
is a cofactor for phosphorylase b, while
it is not required for the activity of phosphorylase a (65”). Interaction of UPN with
inorganic phosphate is indicated by kinetic studies on triose phosphate dehydrogenase (66, 67). The reduction of GSSG by DPNH in the presence of glutathione
c
reductase from yeast is stimulated by phosphate, while the reaction with ‘WNH
is not (57).
It is therefore quite apparent that the cofactors can interact wit-b the enzymes
and with other cofactors in several ways and that a sharp demarcation between the
functions of nucleotides and SH cofactors does not exist. Although it is possible that
in some of the above quoted instances the unexpected role of the cofactors ~nay k-,e
due to an interaction with the protein rather than to a participation in the reaction
mechanism, it appears very likely that in the mechanism of transport of electrons,
phosphates and acyl groups, the nucleotides and SH cofactors have mdtiple rather
than a single function.
Inferaction Between the Cujuctors and the Pre/eins
Warburg (68) has emphasized the role of pyridine nucleotides :t.s prosthetic
groups of enzymes, he refers to ‘pyridinoproteins.’
ObjecCons to this view h:~ve been
January
1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
9
raised by others (38, 69) who look upon pyridine nucleotides as hydrogen acceptors
more alike to a reducible substrate. This difference in viewpoint (besides serving as
a welcome subject for polemic discussions) may actually be a reflection of a dual
physiological role of the nucleotides. These compounds may serve as prosthetic
groups, firmly attached to the enzyme protein, but also as hydrogen carriers freely
dissociable and mobile within the cell. In the latter capacity they may be essential
for oxidation-reduction
reactions catalyzed by soluble enzymes (e.g. in glycolysis),
as well as for the formation of important cellular reductants such as GSH, which
probably plays a major role in the maintenance of the oxidation-reduction
potential
within the cell. On the other hand the firmly bound nucleotide may participate in
functions other than simple oxidation-reductions
as was pointed out above. In
coupled phosphorylation,
the structural proximity of firmly bound nucleotide at
the active center of electron transfer may permit a selective interaction with phosphate with an efficiency which could not be achieved in a solution containing complex and competitive mixtures of enzymes and cofactors.
The existence of a functional division of bound and free nucleotides within the
living cell is indicated by the apparent inability of pyridine nucleotides to penetrate
into the mitochondria. Lehninger and his collaborators have shown (cf. 3) that
although endogenous DPN is readily utilized for coupled phosphorylation
in the
presence of a suitable substrate such as /3 hydroxybutyrate,
added DPNH is not
used in phosphorylation
unless the mitochondria are first exposed to hypotonic
conditions which presumably alter their permeability. If an exclusion of external
DPNH bv mitochondria, which are the site of coupled phosphorylation, takes place
within. the living cell, it is apparent that reduced DPN, formed by soluble enzyme
systems e.g. during glycolysis, is not available for the efficient process of oxidative
phosphoryla tion.
The protein portion of an enzyme without its coenzyme is frequently referred
to as apoenzyme. The implication that the apoenzyme functions only as a carrier
for the functional coenzyme (68) is most certainly erroneous. There is evidence for
the fact that the protein not only plays a role by determining substrate specificity,
but may participate actively in the catalytic process. This is most clearly demonstrated by the formation of acyl enzyme in the case of triose phosphate dehydrogenase which has been freed of DPN (so).
There is a remarkable similarity in the amino acid composition of triose phosphate dehydrogenase from yeast and from rabbit muscle (70). A crystalline protein
was recently isolated from papaya juice and analyzed for amino acids. Its composition was similar to that of egg lysozyme, and a test revealed the crystals to be indeed
1ysozym.e (7 I). The similarity of amino acid composition of enzymes from such
diverse sources as mentioned above is not likely to be accidental and points to
the amino acid sequence as a determining factor in the interaction between the
proteins and their cofactors and substrates. An experimental approach to the specific
site of the interaction between apoenzyme and DPN will be discussed in the section
on triose phosphate dehydrogenase.
The apoenzymes contain a specific and limited number of sites which may bind
several moles of pyridine nucleotides. In triose phosphate dehydrogenase there are
at least three such sites (50) and four in alcohol dehydrogenase (72). The K, values
for the oxidized and reduced nucleotides show considerable variations in different
dehydrogenases and are not always identical with the true dissociation constants
(48, 49, 72, 73). It is of interest to note that the ‘oxidizing enzyme of fermentation’
(triose phosphate dehydrogenase) binds oxidized DPN more firmly than DPNH,
IO
E. RACKER
Volume
35
while in the case of the ‘reducing enzyme’ (alcohol dehydrogenase) the opposite is
the case. Thus the K, values appear to favor the degradation of carbohydrates rather
than the process of synthesis.
The specificity of the interaction between the apoenzyme and the coen.zyme is
usually quite rigid. Many dehydrogenases will react only with DPN and not at all
or sluggishly with TPN or desamino DPN, others will only use TPN as coenzyme.
There are however examples of dehydrogenases which react nearly equally well with
DPN and TPN such as glucose and glutamic dehydrogenase. It is of interest that
the same enzyme from different tissues or organisms may show a different nucleotide
specificity. Glutamic dehydrogenase activity is said to be TPN specific in some
microorganisms (74) but DPN specific in others (75) as well as in plants (76), while
the beef liver enzyme reacts with either. An aldehyde dehydrogenase from liver is
DPN specific (77), while a similar enzyme from yeast acts with TPN as well (78).
Malic enzyme from liver is TPN specific (79) from L. arabinosus DPN specific (80).
Yeasts (61) as well as animal tissues(81) contain two dehydrogenasesfor isocitric
acid, one for TPN, the other for DPN. An interesting example of specificity has been
recently described by Kaplan et aZ. (62) in the caseof pyridine nucleotide transhydrogenase from Pseudomonas jluorescens. The enzyme requires for activity the
presence of an adenosine-2’-phosphatederivative, which can be supplied by adenosine-2’-phosphateitself, or by adenosine-2’)5’-diphosphate or by TPN. The activation by the latter, which actually may also be a reactant in transhydrogenation
reactions, is complicated by the fact that TPN is inhibitory at high concentration
and can only be used at low concentration and in the presenceof phosphate. The
inhibitory effect of TPN can be counteracted by adenosine-2’-phosphate.Another
curious alteration in reactivity of a nucleotide with a protein that might. lend itself
to a study of enzyme mechanismswas reported by Pullman et ul. (82). The authors
observed that with lactic dehydrogenasefrom pig heart, desaminoDPN is lessactive
than DPN for lactate oxidation, but desaminoDPNH is more active than DPNH
for pyruvate reduction. Since these experiments were carried out. at high nucleotide
concentrations they probably represent a true difference in the catalytic activity of
the enzyme.
Although there are claims to the contrary, it is doubtful that nonspecific hydrogen acceptors can replace the pyridine nucleotide in its function wit.h the dehydrogenases.In a frequently quoted paper, Dixon and Zerfas (38) presented carefully controlled experiments indicating the substit,ution of alloxan and other oxidizing
agents for DPN as hydrogen acceptor in alcohol and malic dehydrogenasecatalyzed
reactions. In an attempt to repeat these experiments in our laboratory with cryst,alline alcohol dehydrogenasefrom yeast, it was found that acetaldehyde was formed
from alcohol with alloxan as hydrogen acceptor, but, that the reaction required
traces of DPN in order to proceed at significant rates. In order to demonstrate the
combined effect of both DPN and alloxan on alcohol oxidation it. is essentialin
these experiments to use an alcohol dehydrogenase preparation which contains
neither DPN nor DPNH oxidase (the latter can be found as contaminant in crude
crystalline preparations of ADH). It becameapparent from these and other esperiments t.hat alloxan oxidizes DPNH under the experimental conditions and acted
as the final hydrogen acceptor. These findings fully explain the data of Dixon and
Zerfas provided one assumesthat trace a.mount.s of DPN were present in their
preparations. It should be pointed out h owever tha~t t,he aut.hors considered this
possibility and tested their purified dehydrogenasepreparation for DPN. Employing
January
I 955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
II
a very sensitive methylene blue and flavoprotein-catalyzed
test for DPN, they
failed to discover its presence. However, recent findings of the presence of firmly
bound DPN in crystalline enzymes reopens the question of the validity of their
conclusions, since the flavoprotein catalyzed test may be unsuitable for the detection of protein-bound DPN.
Finally a few words may be said about the specific sites on the proteins which
combine with the pyridine nucleotides. In some instances in which studies in this
direction have been made, SH groups were implicated. This has been indicated by
direct spectrophotometric
measurements (45, 48, 49) or indirectly by studies on the
protective effect of pyridine nucleotides against enzyme inactivation by some oxidizing agents (47) and by more specific ‘SH inhibitors’ such as N-ethyl maleimide
(so). It is clear however that the SH groups do not represent the only point of attachment since in the case of triose phosphate dehydrogenase ‘oxidized enzyme’
contains bound DPN (83) and IAA inactivated enzyme binds DPN quite firmly
(50). An approach to the other sites of binding have been made by Astrachan (84)
who studied the susceptibility of bound DPN to the action of various DPN-degrading enzymes. The ability of DPN fragments to combine with the enzyme was
also investigated, and only adenosine diphosphate ribose and desamino DPN were
found to be bound by the enzyme. The results with DPN-degrading
enzymes were
consistent with the idea that in oxidized enzyme DPN may be bound by its pyrophosphate group.
The specificity of thiol cofactors is usually quite rigid, although exceptions have
been reported (46). In several instances dephospho CoA can substitute for CoA (85)
and in the case of glyoxalase, asparthione and isoglutathione can replace glutathione
(86). Little is known however regarding the role of the pantothenic acid moiety of
CoA or of the peptide linkages of GSH for the catalytic process or for the combination with the enzyme protein.
Mtiltitude
of Dehydrogenases
and Their
Interaction
With Substrates
Multitude of Dehydrogenases. One of the most challenging problems in enzymology concerns the mechanism of substrate activation. In the early days of
enzymology there appears to have been considerable resistance to the notion of a
multitude of enzymes for the various substrates. Since it was considered unlikely
that a minute microorganism such as E. coli could contain a specific enzyme for
every one of the hundreds of substrates which it was capable of utilizing, a challenging theory was proposed to overcome this apparent difficulty (87). It was suggested that the bacterial cell possessed
a limited number of activating centers common to certain groups of substrates. In spite of the fact that the advances of the
past 20 years has increased vastly the number of substrates known to be utilized
by microorganisms,the theory of common activation centers has been abandoned,
mainly beca.useit has become possiblein a large number of substrate activations
to separate the protein catalysts from each other and thus demonstrate their individualities. Indeed, instead of finding common enzymes for similar substrates,
discoveries of multiple enzymes for a single substrate were made. Two distinct and
separableisocitric dehydrogenasesexist in yeast (61) and animal tissues(81). The
liver of someanimals contains three different enzymes catalyzing the oxidation of
acetaldehyde to acetic acid (cf. 88). This latter fact is especially surprising when
one realizes that social activities leading to acetaldehyde formation are not pursued
by most mammals.It is quite possiblethat someof these enzymes act under physio-
12
ILRACRER
Vohme 35
logical conditions on substrates other than acetaldehyde, e.g. long chain aldehydes
or even compoundslike xanthine which is attacked’by one of the aldehyde oxidases.
In liver tissue one can detect two different enzymesoxidizing malic acid (cf. 4), two
alcohol oxidation reactions (cf. 89) and two enzymes which cleave citric acid to
acetyl CoA and oxaloacetic acid (90). In most instancesthe enzymes can be shown
to differ somewhat in their reaction mechanism.The hydrogen carrier may be of a
different oxidation-reduction level as is the case in DPN-linked and flavin linked
aldehyde dehydrogenases.The TPN-linked isocitric dehydrogenasediffers from the
DPN-linked reaction in yeast, the latter showinga marked stimulation by adenosine5’.phosphate. The malic dehydrogenasein liver catalyzes the DPN-linked oxidation
of malic acid to oxaloacetate while the malic enzyme catalyzes a TPN-linked oxidative decarboxylation to pyruvic acid.
Even more striking differencescan be noted when enzymesfrom different sources
are examined. The enzymatic mechanism of activation of acetate to acetyl CoA is
very different in animal tissuescompared to bacteria, which form acetyl phosphate
as an intermediate. The action of acetaldehyde dehydrogenasein animal tissue i.s
again different from that of bacteria which require coenzyme A for the process.In
most instances the reason for these variations in mechanism is not apparent; in
someone might speculate that they fulfill a specific purpose. The role of the ‘malic’
enzyme is probably to furnish malic acid from pyruvate and COZ, while malic dehydrogenaseoxidizes the malic acid, to oxaloacetate. Thus the two enzymes function
in seriesrather than in parallel, catalyzing the synthesisof OAA. The oxidation of
aldehydes in mammalian liver does not need to take the hurdle of an acyl CoA in=
termediate if its ‘purpose’ is that of detoxifying aldehydes. The sacrifice of a single
high energy bond is unimportant in a tissue which can produce over IO ATP by oxidizing the acetic acid which is formed. But in a bacterium which may utilize acetic
acid to make acetaldehyde, which it requires for the formation of desoxyribose-sphosphate, the passagethrough a reducible ‘active acetate’ may represent an enzymatic mechanismvital for survival. Of coursewe realize that such teleological reasoning may be consideredby someto be deplorable.
The Enzyme-Substrate Compound. The major contributions to our understanding of the interaction of enzymes with their substrateshave come from kinetic
and spectroscopic studies. With hemoproteins additional information has been
gained by measurementsof paramagnetic susceptibility. Critical reviews on this
subject have been recently written by Chance (91, 92). While it is apparent t.hat
kinetic data cannot prove a mechanismof enzyme action, the more direct spectrophotometric measurements on enzyme-substrate compounds have the drawback
that more than one compound between substrate and enzyme may be formed, particularly when the substrate is a very reactive substancesuchasH202 or an aldehyde.
Protein-substrate compoundswhich are not involved in the catalysis may be formed,
The recognition of the limiting true enzyme-substrate intermediate, or as Chance
calls it, the ‘Michaelis compound’ must depend on a correlation between its formation and degradation on the one hand, and accurate kinetic measurementsof the
overall catalytic processon the other hand.
Much speculation and calculation has been devoted to the postulated existence
of ternary complexesin systems involving the interaction of a donor and acceptor
component. No direct evidence is yet available for the existence of such complexes
and in a few casesof direct analysis a sequential processinvolving the protein as
carrier between acceptor and donor has been demonstrated (57, 91, 93). Isotope
January rg55
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
I3
studiesmay prove of great value in the determination of the reactionsequence when
several reactants participate (59, 94, 95). It should be emphasizedhowever that the
existenceof numerousexchangereactions make it imperative that enzymes of highest
purity be used in such isotope studies. Preliminary data of this type with partially
purified preparations indicate that high energy enzyme-phosphate or enzymenucleotide bonds can be formed, capable of secondary reaction with suitable acceptors.
Unfortunately only very few data are available demonstrating the formation
of an enzyme-substrate complex by its isolation. These experiments are particularly
difficult to carry out, since they require a> large amounts of enzyme and b) stability
of the intermediate compound. While the first condition can be met with someenzymes which are readily prepared in gram quantities, the secondcondition has to be
met by setting up artificial conditions.
Formation of acylenzyme has been accomplishedin the caseof triose phosphate
dehydrogenaseby two different procedures. Acylenzyme was formed with acetyl
phosphate as substrate. Further enzyme action was prevented by boiling at a PH
which preserved the acyl bond and precipitated the denatured acylenzyme. The
secondand lessdrastic method consistedof depriving the enzyme of its bound DPN
and thus preventing the DPN dependent catalytic degradation of acylenzyme. The
principle in both procedures is essentially the same-it is based on either a permanent or a temporary inactivation of the overall catalytic process,thus permitting
the accumulation of an intermediate enzyme complex (so).
An intermediate enzyme compound was also recently discovered in the case
of phosphoglucomutase(96). It was shown that in the reaction
Glucose-I-phosphate
+ Glucose-I, 6-diphosphatee
Glucose-I, 6-diphosphate+ Glucose-6-phosphate
the enzyme accepts and donates phosphate in a cyclic manner. With glucose-6phosphate or glucose-I-phosphate and large amounts of phosphoenzyme, glucoseI, 6-diphosphate was formed in amounts stoichiometric to the amount of phosphoenzyme. The de-phosphoenzymeformed in this processcould be rephosphorylated
by incubation with glucose-1,6-diphosphate. Thus a similar principle-omission of
the acceptor molecule-permitted stabilization of phosphoenzyme.
Another aspect of enzyme substrate interaction often aids in studies of enzyme
mechanism. Considerable specificity in regard to substrate activation has been
claimed with many enzymes; however, closer examinations revealed in some instancesthat related substratesare attacked slowly if large amounts of enzymes are
used. Thus a slow motion picture of the enzyme, perhaps sometimesslightly distorted, can be obtained. Objections might be raised against the use of large enzyme
concentrations and the use of ‘nonphysiological’ substrate. The possibility of nonspecific chemical interactions between the substrate and the protein, as well as the
occurrence of side reactions due to traces of impurities, makes it indeed essentialto
evaluate such experiments together with data obtained with ‘natural’ substrates.
It is apparent from the above discussionthat neither the kinetic nor the ‘static’
approachescan be regarded as decisive in the elucidation of the interaction between
the enzyme and the substrate. Each of the various approacheswill at best make a
contribution to the circumstantial evidence required to make a case.
It is possibleto account for the optical specificity of enzyme catalyzed reactions
by assumingthree ‘sites’ of attachments for the substrate (97, 98). The concept of
14
E. RACKER
Volume 35
the site is not limited to chemical bonding, but may include electrostatic or van der
Waals forces or the fitting of a substrate group into a ‘hole’ in the protein by the cohesive forces of water (22, 99). Experimental approachesto properties and number
of the anchoring sites of substrates have been made by kinetic studies (100, Iooa,
IOI)
and also by study of the influence of inhibitors and modifications of the substrate on the interaction. The latter approach, which has yielded very valuable in
formation in the caseof hydrolytic enzymes (cf. 102-104)
has not been extensively
explored with dehydrogenases.
TABLE
4
Croup I
R’
R’
R’
I
I
I
CHOH = C=O or CT==0 + CO2
I
I
I
R2
R2
R3
A) Enzymes oxidizing alcohols
C) Enzymes oxidizing hydroxy acids
I) Alcohol dehydrogenases
I) Lactic and malic dehydrogenase and
2) ac-Glycerophosphate dehydrogenase
others
3) Homoserine dehydrogenase
2) ‘Malic’
enzyme, ‘isocitric’ enzyme
and ‘phosphogluconic’ enzyme
B) Enzymes oxidizing or reducing cyclic compounds
3) Hydroxy acid thiol ester dehydroI) Hydroxysteroid dehydrogenases
genases
2) Quinone reductase
0) Enzymes oxidizing hemiacetals
3) Quinic and shikimic dehydrogenases
1) Glucose and glucosed-phosphate de4) Inositol dehydrogenase
hydrogenases
z) Thiohemiacetal dehydrogenases
IT) Formic dehydrogenase
Group II
R1
R1
R1
I
I
I
c=o * C=O or C--O + CO2
I
I
I
R2
S
S
R3
R3
A) Glyceraldehyde+phosphate
dehydrogenase. B) Pyruvic dehydrogenase. C) a ketoglutaric
dehydrogenase. D) /3 aspartyl semialdehyde dehydrogenase.
Group III
R’
R’
I
I
R2CH F= RT
1
II
R3H
R3
A) Amino acid dehydrogenases. B) Dihydroorotic dehydrogenase and others.
Group IV
2RSH e RSSR
A) Glutathione reductase. B) Cystine reductase. C) Lipoic acid dehydrogenase. D) Hydrogenases
and the ‘Hill reaction.’
Groupv
Nucleotide transhydrogenases
A) Pyridine nucleotide transhydrogenases. B) Pyridine-nucleotide
genases.
flavin-nucleotide
transhydro-
January
1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
15
A remarkable stereospecifrcity has been pointed out for a large number of
dehydrogenases (22), to which may be added the example of the apparent nonspecificity of bacterial fermentation leading to D +-lactate. This reaction has been recently shown to be due to the participation of two dehydrogenases, one for L-lactic
the other for n-lactic acid, the latter being removed during purification. The combined action of both enzymes catalyzes a DPN dependent racemization of lactate
(105).
On the other hand the possibility of an enzyme with two points of attachments,
which would catalyze the formation of racemic mixtures cannot be entirely dismissed. It may be profitable to investigate this possibility kinetically in the case of
other racemases which do not require DPN or TPN.
Catalytic Process. Little solid knowledge is available in regard to the actual
mechanism of the catalytic process. A most important development has come from
experiments with labeled substrates (cf. 22, 106, 107). Other fruitful approacheshave
been made by spectrophotometric studies of the interaction of substrate with the
coenzyme-enzyme complex (45, 92), by studies with inhibitors and by kinetic investigations (45, 72, 108). These experiments will be discussedin detail with the
appropriate enzymes.
Models of Enzymes, Coenzymes and Substrates. Important information in
regard to enzyme mechanism has been gained from the use of ‘enzyme models,’
‘coenzyme models’ and ‘substrate models.’ Coenzymes can occasionally serve as
sluggish enzyme models. Oxidation of DPNH by riboflavin phosphates (36), cocarboxylase catalyzed decarboxylations and condensations(109), and chemical transacetylation reactions with acetyl CoA (IO, IIO) can be quoted as examples. The
vitamin portion may serve as model for the coenzyme, e.g pyridoxal in transamination (III)
and N-methyl nicotinamide in oxidation-reductions (15, 16). Lynen has
used SH compoundsas models for coenzyme A (46). Substrate models have played
an important role in specificity studiesof polymer substratessuch as proteins (103).
One of the earliest modelsfor biological oxidations was charcoal in the classical
experiments of Warburg (I I 2). Langenbeck has recently reviewed someof the older
work on enzyme models(I 13). A decisive new development in ‘model enzymology’
has comefrom the concept of polyfunctional catalysis (114). In studies on acid-base
catalyzed mutarotation of tetra methyl glucoseSwain and Brown discovered a high
efficiency of action exhibited by compoundswhich contain two active groups in the
samemolecule. Thus a-hydroxypyridine, a ‘double headedenzyme model,’ containing
an acid and base group is visualized as making a ‘concerted’ attack, which results
in the simultaneousremoval and addition of a proton to the sugar. Extensive kinetic
data indicated the formation of a substrate-catalyst complex not unlike that encountered in enzyme catalyzed reactions.
Classijication
of Dehydrogenases
The specific enzymes which catalyze hydrogen transfer reactions can be classified according to the type of reaction which they catalyze. An attempt at such a
classificationappearsjustified in spite of the fact that in many instancesthe reaction
mechanismis still obscure.It may help to compareenzymes from the point of view of
primary reaction mechanismand thus to avoid confusion created when e.g. pyruvic
dehydrogenaseis listed as a DPN-linked enzyme.
The following five groups of nucleotide-linked dehydrogenaseshave beenselected
for discussion(table 4).
16
Group I catalyzes the reaction type
35
I
RI
(I)
Volume
E. RACKER
Rl
I
CHOH
I
+
I
DPN+(TPN+)
+ C-0
I
R2
+
DPNH(TPNH)
+
H+
R2
or
C=O
I
+CO,
+ DPNH(TPNH)
+
H+
Rt
This group is the largest and includes enzymes which oxidize alcohols, hydroxy acids
and hemiacetals. Enzymes which catalyze the oxidative decarboxylation of hydroxy
acids and of formic acid can be readily fitted into this group until the detailed mechanism is shown to be of another type. Triose phosphate dehydrogenase has been listed
in group II although the possibility of a thiohemiacetal as intermediate in the reaction has not been excluded (57).
Grozcp II contains enzymes which are proposed to operate by an aldehydolysis
of an S---S or an S-C bond according to reaction type 2
In this reaction R2 may be DPN and may become dissociated on reduction as in
glyceraldehyde+phosphate
dehydrogenase, or it may be the sulfur of lipoic acid
and be reduced to SH during pyruvic acid oxidation.
hwp
III contains enzymes which catalyze a process in which a double bond
other than with oxygen is formed following dehydrogenation according to reaction
type 3
RI
R1
I
I
(3) R”---C--H
+ DPN’(TPN+)
s
R2-C
+ DPNH(TPNH)
+ H+
i
RLH
II
R3
This group has been least well studied. The amino acid dehydrogenases have
been classified in this group since experimental evidence does :not favor the nonenzymatic formation of an imino compound (IIS).
Because the mode of action of
these enzymes has not as yet been elucidated their grouping is rather arbitrary.
The best characterized of these reactions is that catalyzed by dihydro erotic dehydrogenase (I 16). The reaction with dihydrofolic acid and DPNH to give tetrahydrofolic acid is now being investigated (I 17).
Group IV comprisesthe enzymes catalyzing either the oxidation of an SH or
the reduction of an S-S compound. In most known instancesthe hydrogen carrier
is a pyridine nucleotide, but in the case of glutathione transhydrogenase GSHGSSG acts as hydrogen carrier system (57). Hydrogenase and the ‘Hill reaction’
may be listed here also although the evidence for SH group participation is only indirect.
Jantiar
y 1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
=7
Gaup v lists the enzymes, which catalyze transhydrogenation
reactions either
from one pyridine nucleotide to another or from a pyridine nucleotide to a flavin
nucleotide. The latter is usually firmly attached to a protein, forming a yellow
enzyme. This group of enzymes is of particular interest in view of the formation of
high energy bonds in the process of coupled phosphorylation.
SOME PROPERTIES
OF INDIVIDUAL
DEHYDROGENASES
No attempt will be made to cover comprehensively all nucleotide-linked dehydrogenases,or even thoselisted in table 3. Instead, a few enzymes which have been
studied more thoroughly will be discussedin greater detail, and somefeatures and
properties of other dehydrogenaseswill be presented. Descriptions of purifications
and assaysof enzyme activity in crude and purified systemswill be entirely omitted,
since they will be dealt with elsewhere(118). Historical aspectsand distribution of
the enzymes will not be covered either, since they have been discussedin other reviews (7, 119).
Group I
Enzymes Oxidizing Alcohols. Alcohol dehydrogenase
from yeast (yeast A Da>.
The discussionof this enzyme has been kept formally separated from that of liver
ADH, becauseof the striking differences between the two proteins, but a complete
separation of the discussionwas neither possiblenor desirable.
Crystalline alcohol dehydrogenasehas been obtained from brewers’ yeast (120)
and from bakers’ yeast (121,
122).
The enzyme catalyzes the reaction: Alcohol +
DPN+Aldehyde + DPNH + H+. In the presenceof small amounts of enzyme
[DPNH] [Acetaldehyde] [H+] =
the equilibrium constant K =
1-q x 10-l?
[DPN] [Ethyl alcohol]
The effect of hydrogen ion concentration- on the equilibrium has been used to
advantage for the determination of DPN in the absenceof carbonyl trapping reagents (I 21). While at an alkaline PH DPN is reduced with alcohol excess,at prr
7.0 with aldehyde excessDPNH is completely oxidized. These changesare quantitatively measuredat 340 rnp in the presenceof crystalline alcohol dehydrogenase.
Electrophoretic analysis of yeast alcohol dehydrogenaserevealed the presenceof
one major and one minor component. Several recrystallizations did not change this
pattern either at PH 5.0 in 0.1: M acetate buffer (122) or at PH 8.0 in Verona1buffer
(40). The smallerand inactive component representedbetween 5 and 20 per cent of
the total protein, depending on the type of preparation used, length of &ly&
etc.
(122).
a) Inactivation studies. The crystalline enzyme can be dialyzed against distilled
water and lyophilized (I 20, I 21). However, this procedure occasionally leads to
considerableenzyme inactivation which can be avoided by dialysis against 0.01 M
buffer at PH 7.0 (40). The enzyme is very sensitive to heavy metals, especially
copper (120) and appears to be rapidly inactivated by contact with rubber stoppers
(I 23). In dilute solution a protective colloid, e.g. serumalbumin, protects the enzyme
against
rapid inactivation (I 2 I).
These findings may account for considerablevariation in the IL., with various
crystalline preparations. Values for the turnover number for alcohol oxidation between 25,ooo-3+00/150,000 gm. protein have been reported (120,
I22),
and even
higher values have been observed (123).
Yeast alcohol dehydrogenaseis an ‘SH-enzyme’ exhibiting marked sensitivity
toward IAA (I 24) and other SH reactive compounds.The enzyme is protected against
1%
E. RACKER
Vdume 35
IAA by the addition of DPN (125, 126). This is in contrast to glyceraldehyde-3-phosphate dehydrogenase(TDH), which becomesmore sensitive to IAA in the presence
of DPN (127, 128). This fact, together with the protection afforded to the enzymes
by the respective substrates (ethanol and glyceraldehyde-3-phosphate) against
IAA (125, 126), permits a differential inactivation of the two enzymes in the presence
of inhibitor (I 26). This feature has been of somepractical usefulnessin phosphorylation studieswith alcohol dehydrogenaseand alcohol as hydrogen donor (3, 130).
b) Specificity. Yeast alcohol dehydrogenasehas beenreported to be specific for
DPN (82). However, Stafford and Vennesland (13 I) have observed TPN reduction
with twice recrystallized ADH, at a rate I/ISOO
that obtained with DPN. Similar
high activity ratios for DPN/TPN have been reported with other dehydrogenases
(132).
It should be pointed out that a TPN-linked alcohol dehydrogenaseis present
in yeast extracts (40). The possibility that crystalline ADH is contaminated with the
TPN enzyme must be considered.The experiments of Dixon and Zerfas (38) on the
substitution of DPN by other oxidizing agents in the alcohol dehydrogenasereaction
have been critically discussedin a preceding section on “Interaction between the
cofactors and proteins.”
Various alcoholscan serveassubstrate for the crystalline yeast enzyme (I 20, I 25).
Ethanol and ally1 alcohol are the most rapidly oxidized substrates,n-propyl alcohol,
jz-butyl alcohol, n-amyl alcohol and isopropyl alcohol are oxidized at progressively
decreasingrates. Considerably higher concentrations of ADH (20- to so-fold) are
required to demonstrate appreciable rates of oxidation with methanol, ethylene
glycol, isobutyl a.lcohol,set-butyl alcohol and glycerol. In connection with the problem of glycerol fermentation, it is significant that both dihydroxyacetone and glyceraldehyde are rapidly reduced at high concentrations in the presence of DPNH
and yeast ADH (40, 133). Studies on the effect of substitutions of the substrate (125)
revealed that NH2CH2CH20H and similar compoundswere not utilized, nor did they
inhibit alcohol oxidation at concentrations five-fold that of ethanol. Substitution
with a halogen however gave rise to inhibitory compounds, e.g. fluoroethanol in
five-fold excessinhibited ethanol oxidation completely. Fluorobutanol was not inhibitory, and in fact was itself slowly oxidized.
The interaction between yeast ADH, coenzyme and substrate is stereospecific.
With deuterium-labeledethanol it wasshown that only one of the two enantiomorphs
is oxidized by the yeast enzyme and that reduction of DPN is stereospecificin regard
to the plane of the nicotinamide ring (cf. I 22).
c) Kinetic studies. Negelein and Wulff in studies on the kinetic properties of
ADH from brewers’ yeast determined the Michaelis constants of the various reactants. No distinction was made between the true dissociation constant (&)
and the Km values (120). In view of the fact that considerable discrepancies between
the K, and X0 values have been reported in various enzyme catalyzed reactions
(cf. 73), Hayes and Velick (122) determined the ADH binding of DPN and DPNH
by the ultracentrifugal separation method. According to their calculat,ion 4 molecules
of DPN or DPNH are bound per molecule of protein and, similar to liver ADH
the reduced and oxidized form of the coenzyme compete with each other (48,49). As
can be seen from table 5 in the case of yeast alcohol dehydrogenase the K,
values and the apparent dissociation constant are not very far apart, DPNH being
bound more firmly than DPN.
The relationship between the equilibrium constant, the Km values and maximum
in which kT
velocities is formulated by: K, = (V~,,,Ka&DPN&‘I/2 max &&,&,
Jamary
x95;
PYRIDINE
TABLE
NUCLEOTIDE-LINKED
5. SOME
PROPERTIES
ENZYMES
OF YEAST
ADH
19
ADH
ADH From
Bakers’ Yeast
From
Brewers’ Yeast
Molecular weight. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Michaelis constants (moles/l. at 26’) (PH 7.6)
K,(DPN)..................................
K,(DPNH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
K,(alcohol).................................
K,(acetaldehyde) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Apparent dissociation constants as determined by
ultracentrifugation at o”-so (moles/l.)
K(DPN)...................................
K(DPNH)..................................
150,~
9
3.0
x
x
2.4
X
1.1
x
1~0,ooo
(49)
Io-5
IO-+
IOIO+
(120)
1.7
2.3
I.8
x
X
x
(122)
IO-4(122)
IO+
IO*
2.6 X IO-~
1.3
X
10~~
representsthe Michaelis constants and VI and V2 the maximal velocities of the forward and back reaction. This equation was discussedseveral years ago (134), without realizing that it had been derived 20 years earlier by Haldane (135). The first
accurate experimental verification for its validity was presented by Negelein and
Wulff (also without reference to Haldane) for ADH from brewers’ yeast (120).
Similar data were obtained for ADH from bakers’ yeast (122). The calculations for
ADH from liver appear to be more complex. According to Alberty (136) the experimental data of Theorell and Bonnichsen (49) are best fitted at several PH units by
the equation: K, = (V?,,,Ka~&PP~~ )/( V&&&&&,
a relation which Alberty
suggestsmay involve an oxidation-reduction of the enzyme protein. A participation
of the SH groups of the protein in the reaction catalyzed by ADH has actually been
suggested(125, 137). Negelein and Wulff (120) reported that with large amounts of
ADH a spontaneousreduction of DPN without addition of substrate takes place,
and alsosuggestedparticipation of SH groups. But becauseof its slownessthe reaction
was consideredunlikely to play a role in the processof alcohol oxidation. Kaplan,
Colowick and Neufeld (138) consider the participation of the enzyme itself in oxidation-reduction unlikely, since ADH did not catalyze an electron exchange between
DPN and desaminoDPN. However, theseexperiments were of necessity carried out
in the absenceof substrate, and therefore do not seemto rule out decisively participation of the protein in the over-all reaction. The direct participation of SH groups
in the hydrogen transfer is however very improbable in view of the experimentswith
deuterium-labeled substrate, since equilibration with water would have been the
result of a formation of labeled SH groups (cf. 22). The latter experiments were
carried out with the yeast enzyme only, and data on deuterium transfer in the case
of liver ADH would be desirable in view of the calculations of Alberty (136).
For both liver and yeast ADH the equilibrium constant has been demonstrated
to be dependent on enzyme concentration (49, 122). This was shownto be due mainly
to the differences in the dissociation constants of oxidized and reduced DPN in the
caseof liver ADH (48, 49). However, the dissociation constants of the enzyme-substrate complex must also play a significant role in determining the equilibrium constant in the presenceof large amounts of enzyme (122, 136). A further complication
arisesfrom observations of Negelein and Wulff (I 20) that brewers’ yeast alcohol dehydrogenase seemsto bind considerable amounts of acetaldehyde nonspecifically,
a finding, which is not unexpected in view of the reactivity of aldehydeswith proteins
in general. If a similar nonspecific binding of acetaldehyde takes place with bakers’
yeast and liver alcohol dehydrogenase,the equilibrium data obtained at high enzyme
concentrations acquire a different meaning. In fact, experiments carried out a number
E. RACKER
20
Volume 35
of years ago (40,121) which indicated an effect of enzyme concentration aswell as of
glycylglycine buffer on K, were interpreted in the light of Negelein and Wulff’s
findings of nonspecific binding. Because of these data, caution was suggestedin
regard to the evaluation of K, values obtained even at low enzyme concentrations.
The agreement between. the observed values for the Michaelis constant and the
dissociationconstant for acetaldehyde-ADH, ascalculated from the equilibrium shift
(122),
has been taken to indicate that the major binding of aldehyde is at the catalytic site. This apparent agreement may be fortuitous however since the Michaelis
constant for acetaldehyde doesnot necessarily represent the true dissociation constant.
d) Mechanism of action. Some clues to the mechanism of action have been
obtained from spectrophotometric studies using enzymes as reactants. In the case
of yeast ADH several investigators searchingfor spectral changes,similar to those
describedfor liver ADH and DPNH, have failed to find them (I 22, I 25). Although
there is general agreement that dramatic spectral changescannot be demonstrated
with yeast ADH, small but reproducible changeshave been observed when DPN
was added to large amounts of ADH (40). The observed increments in absorption
are only partially (35%) prevented by preincubation of the enzyme with IAA under
conditions which lead to nearly complete (95%) inhibition of enzyme activity. Thus
a quantitative correlation between the spectral changeand enzymatic activity similar to that obtained with triose phosphate dehydrogenase(57) dould not be observed
with ADH. The above described absorption changes occur very rapidly and are
completed by the time readings are taken after mixing. It is therefore unlikely that
they are related to the sluggishincrementsof absorption at 340 mp observedby Negelein and Wulff (120).
An interaction between DPN and SH groups of yeast ADH is
also indicated by the protective action of DPN against IAA inhibition (I 25, 126).
Thesefindings point to the formation of a DPN-S-enzyme complex similar to that
observed in the caseof glyceraldehyde-3-phosphate dehydrogenase.In analogy with
the latter, a mechanismof action of ADH may be visualized in which the alcohol substrate cleaves the DPN-enzyme linkage by a processof alcoholysis. There are two
alternative modes of such a cleavage as outlined in the diagram below. In (a), the
hydrogen is transferred directly to DPN to form DPNH, while the rest of the alcohol
combineswith the sulfur to form an enzyme thiohemiacetal, which dissociatesinto
aldehyde and free enzyme. In (b), a DPN-alcohol complex is formed which the enzyme
must cleave in a secondstep to yield DPNH and aldehyde. In view of the isotope
data, it must be assumedthat in both casesthe hydrogen attached to the carbon and
not to the oxygen of alcohol is transferred by the enzyme to DPN.
4
+
H+
+
Enzyme-S-CHOH-CH&--+
b) Enzyme-SH
+
Hf
c--
Enzyme-SH
+
CHS CHO)
+
CH~CHO)
+
DPN >CH~HCH,
(-
<G>
A secondsite for alcohol on the enzyme, independent of the presenceof DPN is
indicated by the fact that ethanol protects the enzyme against IAA in the absenceof
DPN. Similar protective action against IAA by substratesor substrate analogs,which
do not necessarilyreact directly with SH groups, will be discussedin connection with
January
T 955
PYRIDliL’E
NUCLEOTIDE-LINKED
EKZYMES
21
IAA inactivation of triose phosphate dehydrogenases. The previously discussed interaction between DPN and various compounds such as dihydroxyacetone
(32) and
particularly the observations made by Kaplan and his collaborators (139, 140) with
liver alcohol dehydrogenaseand hydroxylamine are in line with the idea of an alcoholysis process.The experiments with hydroxylamine will be discussedbelow.
Alcohol dehydrogenase front liver (liver A DH). The crystalline enzyme has been
isolated from horse liver (141). The enzyme has a molecular weight of 73,000 and
binds 2 molesof DPNH per mole of enzyme; thus per mole of nucleotide the mokular weight is about 37,000, close to the value for yeast alcohol dehydrogenase(I 22)
and for triose phosphate dehydrogenase.The latter enzyme which has a molecular
weight of I 20,ooo
(cf. 72) contains 3 molesof DPN per mole of enzyme (so).
In many other respectsliver ADH is quite different from the yeast enzyme. The
liver enzyme contains lessaromatic amino acids with 280 rnp absorption, it has a
different substrate specificity and a different susceptibility to someinhibitors (e.g.
iodoacetate). Liver ADH is lessthan I/IOO
as active as yeast ADH with ethanol as
substrate, but with acetaldehyde as substrate it is about I/IO as active.
a) Specificity. llliver ADH reacts with TPN at about I/IOO of the rate with DPN
(82). The substrate specificity is broader than that of the enzyme from yeast. The liver
enzyme oxidizes vitamin A to retinene (142, 143).
Ethanol and several higher
primary alcohols are oxidized by both yeast and liver ADH (Kmat PH x0.0 for etha+
no1 = 2 x Io-3, ally1 alcohol = 4 X IO-~, n-propanol = 2.3 X IO-~, n-butanol =
in the caseof liver ADH). The reactivity with methanol doesnot seemto
2.2 x
Io-4
have been clearly establishedwith liver ADH. Theorell and Bonnichsen (49) devoted a paragraph to the important fact that methanol is not oxidized by the crystalline enzyme, while Theorell and Chance (48) in a paper published simultaneously
recorded extensive kinetic data on the reduction of formaldehyde by the sameenzyme
preparation. If it can be established that this extraordinary behavior is due to the
properties of the enzyme, the investigation of this discrepancy might yield important
clues regarding the mechanism of action of the dehydrogenase. The oxidation of
methanol, which seemsto take place quite readily in crude liver preparations, is of
considerableimportance in methanol poisoning. The clinical use of ethanol in these
patients prevents the formation of toxic formaldehyde and permits the urinary
elimination of methanol (144, 145). This therapeutic successhas been quite persuasively explained on the basis of a competitive inhibition of methanol oxidation by
ADH in the presenceof ethanol, a phenomenonwhich has been shown to take place
in Giro with crude preparations of ADH (145).
b) Kinetic studies. Important studies on the kinetics and mechanismof action
of liver ADH have been published by Theorell, Bonnichsen and Chance (48, 49).
They demonstrated a 2oo-fold discrepancy at PH 7.0 between the dissociation constant (&) and the K,, value for the DPNH-enzyme complex. As was shown many
years ago by Michaelis and Menten, and modified by Briggs and Haldane (cf. 135)
the enzyme catalyzed reaction can be written as:
E
+
S 2
ES;
ES
3
E
+
product
k2
k:! + k3
K, = 7
1
The dissociation constant K. for the enzyme-substrate complex (ES) is equal to
the MichaeIis constant K, only when K3is very small compared to k2 ; then K,, =
k 2lk 1 =Ko.
22
E. RACKER
Volz.4nte jy
If however & is small compared to K3, marked discrepanciesbetween the two
values for Km and & arise, and modified calculations are necessary.An analysis of
steady-state kinetics, applied to liver ADH (48), revealed that in the presenceof
excessaldehyde the K, for reduced DPN doesnot dependon the dissociationvelocity
of DPNH but on that of DPN. Thus K, for DPNH could be equal to K, only if the
rates of DPNH and DPN coming off the enzyme were equal. Since in ADH from
liver DPNH is much more firmly bound than DPN, a large discrepancy between
K, and KD results. On the other hand the rates of associationof DPN and DPNH
with the enzyme are rather similar and the tighter bonding with DPNH helps to
explain the greater reactivity of the enzyme with acetaldehyde as compared to
alcohol. In the case of the yeast enzyme the K. values for DPNH and DPN are
considerably closer to each other (122) and the difference between the rates of the
forward and back reaction is lesspronounced (I 20).
The changesin the equilibrium constant at higher enzyme concentrations, first
describedfor liver ADH (4, have been discussedearlier. It was suggestedby Theorell and Bonnichsen that these shifts in equilibrium may play a physiological role,
since the redox potential of the enzyme-bound DPN-DPNH system (& = - 0.21)
as compared to the free nucleotides (EL = -0.28)
is much closer to the level of the
alcohol-aldehyde system (EL = - 0.16). “The reaction velocity in the system would
probably be favored by the diminished potential difference, and the equilibrium
shifted so that the oxidation of ethanol would be favored” (49). This reasoningbecomesapplicable to the open systemof the living cell if the removal of acetaldehyde is
dependent on an enzyme catalyzed reaction which requires a high acetaldehyde
concentration, becauseof a poor affinity of acetaldehyde to the enzyme. It may be
pointed out that this situation actually applies to the enzyme which utilizes acetaldehyde for the synthesis of desoxyribose-s-phosphate(146).
c) Mechanism of action. An interaction between coenzyme nucleotides and the
SH groups of enzymes was first proposedby Rapkine (47). It was also suggestedby
the experiments of Hellerman et al. on the competition between p-chloromercuribenzoate and FAD for D-amino acid oxidase (51). The first quantitative, spectrophotometric measurementson the formation of a complex between -DPNH and liver
ADH were reported by Theorell, Bonnichsen and Chance (48, 49). They found that
the addition of DPNH to crystalline ADH from liver leads to a pronounced shift
in the absorption maximum from 340 1nl-L
for free DPNH to 325 rnp for bound DPNH
(table 6). By the use of an ingeniousmethod specially designedto eliminate changes
due to free DPNH, the reaction kinetics of the ADH-DPNH complex were studied
(48). Accurate titration showedthat at PH 7 there were 2 molesof DPNH bound per
mole of enzyme (K. = IO-~ M at 27’) and at PH IO one mole of DPNH was bound
(K = 3 x Io-6 M). Measurements of various velocities are summarized in the
foliwing formulation of the mode of action of the dehydrogenase(147).
ADH
H+
$-
+
ADH-DP)NH
(
DPNH
kl
=
4
X
k2 =
+
10+bf-~
_____..___~X
sec.+
ADH---DPNH
+
0.4 sec.-l
acetaldehyde
+k4
---------*‘*
’
*“-“M-’
’ __-. sec*-L--..
ADH--DPN+
ADH-DPN+
+-.----k5 =
ka =
2 x
45 sec.+
IO*M-'
x
-------
sec.-l
ADH
+
+
DPN+
ethanol
.hIPU.Wy
PYRIDINE
1955
TABLE
mp:
FreeDPNH..
BoundDPNH
6. MILLIMOLAR
...
. .. .
NUCLEOTIDE-LINKED
EXTINCTION
ENZYMES
COEFFICIENTS
FOR
FREE AND ADH-BOUND
3x0
3.6
315
4.1
320
4.8
325
5.3
328
5.65
330
5.8
335
6.1
5.1
5.5
5.7
5.8
5.65
$6
593
340
6.25
4-7
DPNFI (49)
345
6.1
4-I
350
5.7
3.3
The data provide evidence for the participation of the DPN-enzyme complex in
the reaction catalyzed by the enzyme. The agreement of the values for ,&I and h
either measured directly or calculated from the over-all activity is cited as demonstration of the operation of the (modified) Michaelis-Menten mechanism in a nucleotide-linked dehydrogenase reaction.
p-Chloromercuribenzoate,
which has been shown to inhibit liver ADH activity
(cf. 49), was found to abolish the DPNH shift due to combination with enzyme,
while iodoacetate had neither inhibitory activity nor did it affect the DPNH-ADH
absorption. This correlation between enzyme activity inhibition and in teraction with
the complex is an additional indication for the participation of the latter in the enzyme
catalyzed reaction.
A significant observation was recently reported by Kaplan (140) on the effect of
hydroxylamine, which wasfound to be a competitive inhibitor of liver and yeast alcohol dehydrogenase(139). In the caseof the liver enzyme a tightly bound complex
between DPN, hydroxylamine and ADH is formed which results in inhibition of
enzyme activity. The inhibitory complex can be dissociatedby prolonged dialysis,
which restores enzyme activity. Of specia#linterest are the observations that the
complex exhibits an absorption with a maximum at 300 rnp; that 2 moles of DPN
are required per mole of enzyme to give maximum absorption; and that p-chloromercuribenzoate abolishes the absorption due to the complex. The complex was
found tobe bound more firmly than even DPNH, in line with the fact that aldehyde
reduction is inhibited also. In view of these findings Kaplan suggeststhat hydroxylamine as well asethanol interacts with DPN in the initial step, thus favoring alternative (b) of the above proposed mechanismof alcoholysis.
Alcohol dehydrogenases from other sources.Alcohol dehydrogenasehasbeen found
in various plants but these dehydrogenaseshave not been extensively studied. The
wheat germ enzyme is similar to the yeast enzyme in regard to IAA sensitivity (131).
The reactivity of crude wheat germ extracts with TPN suggestseither a lack of
nucleotide specificity or the presenceof a second,TPN-linked alcohol dehydrogenase.
The occurrence of a TPN-linked alcohol dehydrogenasemay permit the anaerobic
function of the so-called‘oxidative pathway’ of glucose-6-phosphateutilization by
a TPN-linked oxidation-reduction.
Glycerol dehydrogenases. As has been pointed out previously, yeast alcohol
dehydrogenase reacts with dihydroxyacetone, glyceraldehyde or glycerol as substrates (133,40).
The existenceof a separateglycerol dehydrogenasehas beendemonstrated by means of hydroxylamine which inhibits alcohol dehydrogenasebut not
glycerol dehydrogenase (148). The enzyme was found in extracts of Aerobacter
aerogenes and catalyzes the DPN-linked oxidation of glycerol. Neither a nor @glycerophosphates are acted upon. With reduced DPN glyceraldehyde as well as
DHA is utilized, the reaction with the latter being faster. The presenceof a specific
dehydrogenasefor butylene glycol in extracts from the samebacteria (149) opensup
the question of the possibleidentity of the two enzymes. The reduction of glyceraldehydeby DPNH hasbeenobserved with rat liver (150) aswell with beef, pig and horse
liver preparation (40). Since the reaction is alsocatalyzed by three times recrystallized
Volume 35
E. RACKER
24
liver ADH (40), no evidence for a specific liver glycerol dehydrogenase is as yet available.
A glycerol dehydrogenase preparation has been obtained from E. coli (I 5 I).
This preparation catalyzes the formation of dihydroxyacetone
in the presence of
glycerol and DPN. The enzyme has a p13 optimum of IO, similar to t.hat for alcohol
oxidation by liver ADH, and is similarly inhibited at low concentrations of p-chloromercuribenzoate (10-6 M). It is very sensitive to Zn*, Cu* and Fe++. The enzyme
has been purified about q-to 2o-fold and showed no activity with other alcohols
tested (‘ethanol, a-glycerophosphate,
erythritol, D-sorbitol and- D-mannitol). The K,
for DPh was found to be 2.6 X IO--~ M and for glycerol IO-~ M. The enzyme withstood heating for prolonged periods at 6o”, a feature used in the purification procedure.
A DPN-linked enzyme which oxidizes D-sorbitol and L-iditol to the corresponding ketosugars (D-fructose and L-sorbose) was partially purified from rat liver (152).
The K, forsorbitol is 7 X IO-~ and K, is 0.24 at 20' and PH 810.The enzyme4showed
no activity with D-iditol, D-mannitol and dulcitol. Provided that the activity with the
two polyalcohols is due to the same enzyme, the specificity is determined by the
configuration at carbon 2 and 4, but not at carbon 5.
Other dehydrogenaseswhich act on phosphorylated polyalcohols have been.
described but have not been studied in detail (153, 154, q4a).
cr-glycerophosphate
dehydrogeuase (arGDH). This enzyme catalyzes the reversible
oxidation of L-cu-glycerophosphateto dihydroxyacetone phosphate:
CH20HCHOHCH20POaH2 + DPN+ z$ CH20HCOCHzOPOgH2 + DPKH + H+.
Baranowski has obtained the enzyme in crystalline form by refractionating
crystalline Myogen A (155). This remarkable feat of slow fractional crystallization of
a ’homogenousprotein’ into fractions of different enzymatic activities should be
emphasizedfrom the viewpoint of protein ‘purity’ aswell asa procedure for purification. The principle of slow, automatic additions of ammonium sulfate hasbeen systematically applied by Biicher and his collaborators for the separation of a number
of glycolytic enzymes from rabbit muscle (156). ar-G1ycerophosphat.edehydrogenaseis among the enzymes obtained in crystalline form.
a! GDH obtained by Baranowski (155) was found to be very unstablein high dilutions. Highly purified preparations of a glycerophosphate dehydrogenaseobtained
by an unpubl.ishedprocedure were also found to be unstable on dilution, but could
4 A further
study by Edson and his collaborators
(Biochem.
of this reaction revealed that the enzyme catalyzes the oxidation
with a configuration
of I and II.
CHzOH
I
HCOH
CH20H
I
HCOH
HOCH
HCOH
I
and
J. 5 7: 518, 19543 on the specificity
at the second carbon of polyalcohols
II
It is of interest to note that the phosphorylated
deri vatives of the ketoi)entoie,
ketohep tose produced
by this dehydrogenase
serv ‘e as substrates
for transketolase.
ketohexose
January
I95
j
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
25
be protected by the addition of buffered bovine serum albumin (40). In contrast to
these findings, the enzyme of Beisenherz et al. (156) was very stable in dilute solutions. This fact was confirmed in our laboratory with preparations kindly sent by
Dr. Biicher, and their preparative method should therefore be considered the most
suitable for obtaining stable preparations.
The turnover number of a! GDH was found to be 26,500 moles of dihydroxyacetone phosphate per 100,ooo gm. of crystalline enzyme at PH 7.0 at 20'. The rate was
doubled at 30’ (155). The equilibrium of the reaction was measured only at PH 7.0
[Dihydroxyacetone-p]
[DPNH] [H-t]
and from the data the estimated value for K =
[a! glycerophosphate] [DPN]
=
7.2 X IO+*. This value is not far from that obtained for the oxidation of ethanol
by ADH (49, 121).
The ultraviolet absorption spectrum of a! GDH was found to give a low ratio
at 280/260 rnp and the possibility of bound DPN was suggested (155, 156). Recent
investigationsof B&her (157) haveestablished that thenucleotidein a! GDH isneither
DPN nor TPN, and is not reducible by sodium hydrosulfite. The possibility that a
nonreducible nucleotide is essential for CIIGDH may be considered. Such a requirement
would not be unlike the dependence of isocitric dehydrogenase in yeast (61) on
adenosine-5’-phosphate.
Little is known about the specificity of cy GDH. Crystalline Myogen A preparations, which are a rich source for a! GDH, have been shown to be inactive with ,8glycerophosphate, but catalyze the oxidation of I, 2-propandiol-r-phosphate
with
DPN as hydrogen acceptor (q8).
The product has not been identified but by analogy
might be expected to be monohydroxyacetone
phosphate. It has shown that the
product of I, 2-propandiol-r-phosphate
dehydrogenation was oxidized further by
crystalline triose phosphate dehydrogenase. In view of the presence of triose phosphate isomerase and other impurities in Myogen A (50, 158) the nature of the secondary oxidation remains obscure.
Humoserine dehydrogemzse. Cell free extracts from bakers’ yeast have beenfound
to catalyze the reversible oxidation of L-homoserineto aspartic-p-semialdehyde(T59).
CH20HCH2CH NH2COOH + DPN+(TPN+) e CHOC?&CH NHzCOOH +
DPNH(TPNH) + H’. Th e enzyme has been measuredwith DPNH or TPNH and
aspartic+semialdehyde as substrate. Reversibility with L-homoserinebut not with
D-homoserinehas been demonstrated.
‘Chol&e oxidase.’In the course of choline oxidation in liver betaine aldehyde is
formed (160). The oxidation of betaine aldehyde is DPN-linked (161) and will be
discussedtogether with other aldehyde oxidizing enzyme. Claims have been made
that the oxidation of choline to betaine aldehyde is alsoDPN-linked (162). The data
presented to support this conclusionare not convincing, sincethe possibility that the
small stimulation observed on addition of DPN is due to residual aldehyde dehydrogenaseactivitv in the liver particles has not been rigidly ruled out. No DPN
stimulation of choline oxidase activity has been reported by others (163, 164).
Choline oxidasesolubilized with sodium choleate and purified 13.fold did not require
DPN (165).
Enzymes Oxidizing or Reducing Cyclic Compounds. Hydroxysteroid
dehydrogejzases. Mamoli and Vercellone (166) demonstrated the transformation of 4androstene3 I 17-dione to testosterone by fermenting yeast. The first indication, of
DPN participation in thesereactionswas obtained by Samuelset al. (167) who demonstrated a stimulation of the formation of IT-ketosteroids from testosterone in liver
26
E. RACKER
I~rot24me
35
mince. The enzyme responsible for this reaction was later purified from steer liver
(168). A DPN-dependent,
a estradiol inactivating system in rat liver has been described (169). Participation of DPN in the C-I I-fl-hydroxylation
of desoxycorticosterone to corticosterone by adrenal homogenates has also been implicated (I 70, I 7 I).
A dehydrogenationat C&Ill or at Cll---Cl2 wassuggestedas the enyzmatic mechanism
of hydroxylation, but recent evidence does not favor this pathway (172). Several
other systemsof steroid interconversion appear to be stimulated by DPN or TP?G
b73-I7@*
An adaptive enzyme has beenobtained from Pseudomonascellsgrown on testosterone (177, 178). With partially purified enzyme the testosterone oxidation was
[4-androstene-3-,I7-dione] [DPNH]
~- [H+] =
studied and was found to have a K =
[Testosterone] [DPN]
3.6 X IO? At PH 9.0 the enzyme could be usedfor assayof DPN and of testosterone.
The enzyme wasfound to be unstable in dilute alkaline solution, and DPN stabilized
it under theseconditions, SH compoundsdid not. p-Chloromercuribenzoate inhibited
markedly at IO-~ M and DPN again protected completely. An interesting phenomenon
of specificity was observed in regard to protection against alkaline inactivation.
Testosteronedid not protect but 17 P-estradiol, which is alsoa substrate, did protect.
A number of 3 P- and I 7 @-hydroxysteroidswere oxidized by the enzyme preparation and DPN. The adrenal cortical hormones (desoxycorticosterone, cortisone and
17-hydroxycortisone) were inactive. a! Hydroxysteroids were not oxidized by the
purified enzyme, but a separable enzyme specific for 3 CYhydroxysteroids has been
demonstrated (179).
I 7 a-Estradiol and diethylstilbestrol were found to be potent
inhibitors of the P-hydroxysteroid dehydrogenase.Attempts to dissociatethe 3- and
17-OH oxidizing,activity were unsuccessfulin the caseof the bacterial enzyme, but
apparently two different enzymes are present in mammalian tissues(180).
Q&one reductase. An enzyme, partially purified from peas, (18 I) was shown to
accelerate the reaction, DPNH + H+ + p-quinone -3 DPN+ + hydroquinone. For
all practical purposes the reaction is irreversible. The rate with TPNH was about
half that with DPNH. Several derivatives of benzoquinone and napht hoquinone
served as hydrogen acceptor instead of quinone. The enzyme was shown to have a
~JXoptimum at PH 6.5 while the quite rapid, nonenzymatic reaction was unaffected
by per. Of particular interest is the inhibition of the enzyme-catalyzed reaction byI
2 ,4-dinitrophenol and related compounds.
Quiutic and shikimic dehydrogenases. Extracts of A. aerogelzes were found to
contain a DPN-linked enzyme catalyzing the oxidation of quinic acid to 5-dehydroquinic acid (182)
and a TPN-linked enzyme which catalyzes the reduction of 5dehydroshikimic acid to shikimic acid (183). The latter enzyme was demonstrated
also in E. coli extracts.
Inositol dehydrogenase. Extracts from A. aerogetzes grown in the presenceof
myo-inositol were found to catalyze the reversible oxidation of jnyo-inositol to 2
keto-(myo-inositol in the presenceof DPN (184). Myo-inositol + DPN+ $ 2 ketomyo-inositol + DPNH + H+. The equilibrium of the reaction was found to be far
to the left. TPN was inactive.
Enzymes Oxidizing Hydroxy Acids. Lactic and malic dehydrogenases and ofhers.
a) Lactic dehydrogenase (LDH). LDH catalyzes the reaction: r,-lactate + DPN+ e
pyruvate + DPNH + H +. At low enzyme concentration. the value of K =
Pyruvatel WPNHI fH’1 = 4 x Io-12 (121, 185). The reaction with pig heart LDH
[Lactate] [DPN+]
proceedswith TPN also, but the rate with DPN is about 200 times a,srapid (132).
January
1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
27
Crystalline preparations of LDH have been obtained from several sources.Most
extensive studies have been carried out with the enzyme from heart muscle (186)
and the rat liver enzyme (187). The enzyme has been widely usedfor linked assay
systems, e.g. for determination of pyruvate kinase. For this purposeLDH of highest
purity is required. The preparation of rabbit muscleLDH (156) fulfills this purpose,
particularly if recrystallized several times (so). LDH from heart muscle consistsof
two active components (185) which are separableby electrophoresis.Rat liver LDH
is electrophoretically homogeneousat several PH values. The molecular weight
of the
two enzymes is 135,ooo for muscleLDH (185) and 126,000
for the rat liver enzyme
(187).
An amino acid analysis of the latter revealed a high content of leucine, valine
and isoleucine.
SPECIFICITY.
Pig heart LDH was shown to react with a large number of a! keto
and a,y-diketo acids and DPNH (188). Hydroxypyruvate was also found to be reduced (189). Differences in PH optimum and Km values were observed with different
substrates, the K, value for pyruvate being the lowest (K, = 5 X IO-~). The Km
values for other substrates are some IO to IOO times larger. The K, values appear
to vary with enzymes from different sources; a value for Km pyruvate of 9 X 10-6
with rat muscle LDH was reported (187).
Km lactate = IO-~ M with pig heart muscle (185) and over IO times as high with rat liver LDH (187).
INHIBITORS.
Hydroxymalonate has been shown to inhibit lactic acid oxidation
in tissue slices(190). Crystalline LDH was found to be markedly inhibited by a ,ydiketo valeric acid (188). Neither IAA nor p-chloromercuribenzoate inhibited the
enzyme at PH 10.0 (185) and the conclusion was drawn that LDH probably doesnot
depend on the presenceof SH groups. Although this appears reasonablein view of
the rather high concentrations (IO-~ M) of inhibitor used, further investigations at
lower PH values and with different SH inhibitors seemsto be indicated” sincea remarkable variation in susceptibility to various inhibitors exists in dehydrogenases(191).
KINETIC
STUDIES.
The values for the turnover number of various LDH preparations differ somewhat, mainly becauseof differencesin experimental assayconditions
(187).
At comparableconditions pig heart LDH (185) and rat liver LDH (187) have
similar turnover numbers. The maximum value is 32,900 moles of DPNH oxidized
per minute per mole of enzyme at 24’ in Verona1buffer PH 8.8. Emphasishas been
placed on the concentration of the secondreactant in determinations of K, in reactions involving more than one substrate or a substrate and a coenzyme (187).
The equilibrium constant was increased up to 3o-fold by increasing the concentration of LDH (185). The significance of this phenomenon has been.discussed
for ADH (seesection, “Alcohol dehydrogenasefrom yeast”). Chance and Neilands
(192)
have observed spectral shifts with LDH, analogousto those with ADH. The
LDH-DPNH complex is however more difficult to detect spectrophotometrically
becauseof the larger value for the apparent dissociationconstant. Of specialinterest
is the speculation of the authors on the relationship of the large dissociationconstant
to the high turnover number and the probability of not detecting spectral changes.A
broader searchfor this type of correlation. which fits the caseof the liver ADH and
yeast ADH seemsto be indicated. At first inspection it does not seemto hold for
glucose dehydrogenase (193)
or glutamic dehydrogenase (194)
but in view of the
greater complexity of thesereactions a more detailed analysis must be awaited. The
spectral shift due to LDH-DPNH complex which showsa 330 rnp peak is not prevented by addition of p-chloromercuribenzoate.5
___
----ported
5 Since this was written,
an interaction
(NEILA~VDS, J. B. J. Biol. Chem. 208:
between p-chloromercuribenzoate
1954).
225,
and LDH
was re-
E. RACKER
28
Volzcme 35
Experiments with deuterium (195) show that, as in the case of ADH:, there is a
direct and reversible transfer of hydrogen from the substrate to the coenzyme in
the presence of the enzyme and that ADH and LDH remove or add hydrogen to the
same side of the nicotinamide plane.
b) Malic dehydrogenase
(MDH).
This enzyme catalyzes the reaction: I,malate + DPN e Oxaloacetate + DPNH + H+.
K = [~~~laoa~etate~~DPN~l
[Malate][DPN+]
EH+1 _ , 5 x Io-la(52)
’
The equilibrium constant was about doubled when the malate concentration
was raised from 5 X IO-~ M to 5 X IO-~ M, a finding which has been interpreted as
an effect of the ionic strength on the activity coefficients of the reactants (52). Other
salts added to 5 X IO-~M malate also increased the K values.
TPN substitutes for DPN, but the rate with DPN is about 30 times more rapid
(132).
No substrate except malate has been shown to be oxidized by MDH. Straub
has‘ purified the enzyme from pig heart muscle (196). A rather heat stable malic
dehydrogenase has been reported to be present in extracts from a thermophilic
bacterium (197).
Nicotinic acid amide acts as a competitive inhibitor for DPN with
purified MDH (198). Inhibition was also observed with adenine, adenosine and ATP
(199,
zoo). The muscle enzyme and MDH from the thermophilic bacterium have a
Km = 0.01 M. p-Chloromercuribenzoate
has been reported to be highly inhibitory to
MDH, while iodoacetamide was without effect (191).
c) Glycolic dehydrogenase (glyoxylic reductase). An enzyme obtained from
spinach leaves (201) has been shown to catalyze the reaction: glycolate + DPN+ z@
glpoxyl.ate + DPNH + H+. The equilibrium
of this reaction is far to the left,
[Glyoxylate]
[DPNH]
[H+]
k’=
_.-- __._
---~__
= 1.75 X 10-l~ (202).
Large amounts of glycolate
[ Glycolate] [DPN]
(0.5~)
had to be used to demonstrate reversibility.
SPECIFICITY.
Crude enzyme preparations were found to react with both DPNH
and TPNH, but highly purified preparations (over rooo-fold) reacted with DPNH
only. The purified enzyme still contains D-glyceric dehydrogenase. The activation of
glyceric and glycolic acid, however, does not appear to be due to the same enzyme,
because of a) different activity ratio obtained during fractionations b) different susceptibility to p-chloromercuribenzoate
c) summation of activity in the presence of
both substrates at saturating concentrations. The enzyme does not react with pyruvate, acetaldehyde, D-tartrate and mesoxalate (202).
p-Chloromercuribenzoate
(IO-~
M) inhibited the enzyme 68 per cent. Fluoroacetate
( roWi! M), cyanide ( xoW3M and
formaldehyde (IO-~ M) had little or no effect (202).
It has been suggested that glycolic dehydrogenase together with the Aavoprotein
enzyme glycolic oxidase may play a role in the terminal oxidation in plants according to the following mechanism (201):
(I) Substrate
@)
DPNH
+
+
DPN+
Hf
+
Dehydrogenases
>
oxidized substrate
glyoxylic
a--id
+-
G1yoxylic
DPN+
(,;;) Glycolic acid
+
02
Glycolic
--------+
oxidase
DPhTH+
+
H
reducE-E+
+
glyoxylic acid
glycolic acid
+
H2 O2
January
PYRIDINE
q;j
(4) Hz02
=
Sum: Substrate
NUCLEOTIDE-LINKED
Hz0
+
x0,
+
ENZYMES
29
go,
DPN, g’YO’YliC “‘d_,
oxidized substrate
d) Glyceric dehydrogenase
(hydroxypyruvate
reductase). Stafford et al. (203)
have demonstrated the presence of an enzyme in extracts from plant leaves which
catalyze the reaction, D-glycerate + DPN+ s Hydroxypyruvate
+ DPNH + IF.
The enzyme is inactive with either D or L lactic acid and, as pointed out above,
appears to be distinct from the glycolic dehydrogenase (202).
TPNH did not substitute for DPNH with D-glyceric dehydrogenase.
Crystalline lactic dehydrogenase from pig heart has been shown to reduce
hydroxypyruvate
(189). The muscle enzyme is specific for the L-isomers of the hydroxy acids and does not react with D-lactic or D-glyceric acid.
e) P-hydroxybutyric
dehydrogenase.
Although p-hydroxybutyric
dehydrogenase was one of the very first dehydrogenases obtained in cell free extracts prepared
from liver (204) the enzyme has not been extensively studied. The reaction catalyzed
is DPN specific: L-hydroxybutyrate
+ DPN s acetoacetate + DPNH + H+.
The enzyme is specific for the L-isomer. It does not react with p-hydroxypropionic,
or-hydroxybutyric
and y-hydroxybutyric
acid (205). Some of the higher P-hydroxy
acids were found to be oxidized slowly (206) but in view of the crude nature of the
preparation other enzymes may have been responsible for these effects.
f) Diketosuccinic
reductase. Various plant extracts have been found to catalyze the oxidation of DPNH on addition of diketosuccinate (207). The product of
this reduction has not been identified but is suggested to be a! keto-P-hydroxy
succinic acid or its enediol, dihydroxyfumaric
acid. This suggestion is supported by the
ability of the product to reduce 2,6=chlorophenolindophenol.
‘Malic'
enzyme,
‘isocitric’ enzyme arcd ‘phosphoglticoutic’ enzyme. a) ‘MaW
enzyme (M.E.). Inanimal tissues, plants and bacteria an enzyme can be demonstrated
which catalyzes the reaction: L-malate + TPN(DPN)
e pyruvate + CO2 + TPNH(DPNH) + H+.
Extensive reviews on these enzymes have appeared (208, 209). M.E. from wheat
germ (210) is free of lactic dehydrogenase, while the DPN-linked
L. a&itiosus
enzyme has not been separated from lactic dehydrogenase. Therefore, the product
of the reaction in this case is actually not pyruvic acid, but lactic acid. That the
latter does not arise by direct decarboxylation of malate is indicated by the absolute
requirement for DPN for malate decarboxylation. DPN is not needed for the decarboxylation of OAA catalyzed by the same enzyme. OAA-carboxylase
and M.E.
activity were compared in various fractions during purification of M.E. from various
sources and the activities were found to run parallel. In the case of the bacterial enzyme adaptation in the presence of malate gives rise to the simultaneous appearance
of both activities. It should be stressed that the two activities are about equal only
when tested at their respective PH optima (PH 7.5 for WE. and PH 4.5 for OAA
carboxylase). At PH 7.4 the rate of OAA decarboxylation is very small compared
to the rate of malate decarboxylation. Moreover the decarboxylation of OAA can be
inhibited by various substrate analogs, e.g. malonate which do not affect malate
decarboxylation (79, 211, 212).
An important observation has been made with highly purified preparations of
L. arabinosus enzyme. As mentioned previously, lactic acid has been demonstrated
as the end product. Since LDH can be obtained free of M.E. by fractionation or
30
E. RACKER
Volwne
3j
by using unadapted cells it has been assumed that M.E. is a separable enzyme and
pyruvi.c acid is the primary dissociable product of the reaction. In fact it was possible
to demonstrate formation of some pyruvate during the reaction. However, isotope
studies have made it apparent that pyruvate does not necessarily equilibrate with
pyruvate added to the reaction mixture (105) and lactic acid of a specific activity
much higher than that of pyruvate was obtained. Thus the possibility of an M.E.LDH complex which permits the efficient conversion of malic acid to lactic acid is
suggested.
MECHANISIVI OF ACTION. It has been suggestedthat the enzyme is a ‘double
headed’ enzyme containing an oxidative and a decarboxylating center on the same
protein molecule. This concept has received strong support from the following experimental observations: a) Extensive purification failed to indicate separation of the
two activities. b) A combination of highly purified preparations of malic dehydrogenaseand OAA carboxylase from iK Zysodeicticus did not catalyze the reductive carboxylation of pyruvate to malate although at high concentrations the forward re-,
action, oxidative decarboxylation of malate, was catalyzed. c) Other enzymes with
‘multiple heads’ have been obtained which have been shown to be homogeneous
proteins.
Hlowever, the true intermediate of the reaction is unknown. Experimental
evidence, consisting of the lack of reduction of OAA by M.E. in the presence of
TPNH and the absenceof radioactivity in OAA when added as carrier during fixation
of Cl402 into malate, argues against OAA as the true intermediate (cf. 208,
2x3).
Therefore Ochoa and his collaborators have proposed the following alternative
hypothesis, suggestingan oxalacetic hydrate lactone as an intermediate, which is
decarboxylated to enol-pyruvate.
0
OH
I!
I
C-CHz-C-COOH
I
I
0 p-iq
--
+ TPN+ e
0
OH
II
!
C-.-CH2-m-C--COOH
IL--o--I
i
CO2 +
+ TP:SH + H+
u
CHz= C(OH).-COOH
As an alternative hypothesis, it might be suggestedthat the true intermediate
is a product of the cleavage of a TPN-enzyme complex according to a mechanism
similar to that proposedabove for alcohol dehydrogenase.Thus either a malate-TPN
or a SH-malate compound would be formed. It is of interest in this connection that
M.E. is very sensitive to mercurial inhibitors (213a).
The previously mentioned experiments with L. arabinosus enzyme, which demonstrated that during lactate formation from pyruvate added pyruvate did not. equilibrate with ‘enzyme bound pyruvate,’ suggestthe possibility of a similar situation in
the case of OAA. However, the lack of OAA reduction by TPNH and ME. still
disfavors OAA as intermediate. It shouldbe pointed out that, if OAA is not the intermediate, the arguments derived from experiments with combined MDH and OAA
decarboxylase quoted above lose someof their cogency. In other words, it is still
conceivable that a combination of two appropriate enzymes acting on the true intermediates may catalyze the reductive carboxylation of pyruvate. Such experimental
duplication of the action of a double-headedenzyme by two separableenzymes (aldehyde dehydrogenase and transacetylase) has been discussedin the case of triose
January 1955
PYRIDINE
NUCLEOTID~-LINKED
ENZYMES
31
phosphate dehydrogenase (93). Since a reductive carboxylation of phosphoenolpyruvate (PEP) to malate has been demonstrated (214), it should be possibleto imitate ‘malic’ enzyme by the following seriesof reactions.
(I) pyruvate + ATP e phosphoenolpyruvate (PEP) + ADP
(2) ADP + ITP e ATP + IDP
(3) PEP + IDP + COee OAA + ITP
(4) OAA + DPNH + H++ malate + DPN
Since, in a system which rapidly regeneratesATP and which has a high ATP/
ADP ratio, large amounts of phosphoenolpyruvate can be formed (so), the reductive
carboxylation of pyruvate to malate should readily take place. In fact the above series
of reactions is probably responsiblefor the earlier data of Utter et al. (215) and of
Kaltenbach and Kalnitsky (216) on the synthesis of OAA from pyruvate.
b) ‘Isocitric’ enzyme (isocitric dehydrogenase). TPN-LINKED
ISOCITRIC
ENZYME.
The enzyme isolated from animal tissues (217) catalyzes the reversible oxidative
Mn++
decarboxylation of n-isocitrate to cyketoglutarate. D-isocitrate + TPNf $ cxketoglutarate + CO2 + TPNH + Hf.
The reaction has been assumedto go through oxalosuccinate (OSA) as an
intermediate, but similar to the reaction catalyzed by ‘malic’ enzyme, a) the dehydrogenaseand carboxylase have not been separated b) OSA has not been established
as an intermediate of isocitrate dehydrogenation. However, in contrast to WE.,
isocitric dehydrogenasecatalyzes the reduction of OSA with TPNH.
Reversibility of the over-all reaction was demonstrated spectrophotometricallv
as well as by a coupled oxidation-reduction reaction. By regenerating TPNH with
glucose-6-phosphatein the presence of glucose-6-phosphate dehydrogenase the
reductive carboxylation of a! ketoglutarate to isocitrate occurred as follows:
Glucose-6-phosphate
+
TPN+
-+
6-phosphogluconate
a! ketoglutarate +
CO2 +
TPNH
+
+
TPNH
+
H+
H+ -+ D-isocitrate + TPNf
_-Sum: Glucose-6-phosphate + a! ketoglutarate + C O2 --+ 6-phosphogluconate +
D-isocitrate
If measuredin bicarbonate buffer the two acid groups which are formed in the
reaction release2 CO2 while only I CO2 is fixed. Thus by this ingeniousmethod the
rate of CO2 fixation is measuredby the CO2 liberated.
The kinetics and specificity of the decarboxylation of oxalosuccinate have been
extensively reviewed by Ochoa (208) and will not be discussedhere. It may be mentioned that the great specificity and the high affinity of the enzyme for TPN and
D-isocitric acid make it a very suitable tool for the specific determination of these
components. Lotspeich and Peters (218) have recently investigated the sulfhydryl
properties of the enzyme. Monosubstituted arsenicalswere inactive, but diphenylchlorarsine was found to be inhibitory. Iodoacetate was without effect, but pchloromercuribenzoate, which appears to be the most generally effective SH inhibitor, was toxic. This type of rather specific distribution of susceptibility is very
much in line with the observations of Barron and Singer (191) and emphasizesthe
points stressedby theseauthors.
DPN-LINKED
ISOCITRIC
ENZYME.
A DPN specific enzyme which catalyzes the
oxidative decarboxylation of isocitric acid to LYketoglutarate was found in bakers?
32
l-3. RACKER
I~‘Olm2t335
and brewers’ yeast, which contained also the TPN-linked enzyme (61). The DPNlinked enzyme required catalytic amounts of adenosine-5’.phosphate,which was
found to have a high affinity for the enzyme (Km = IO-~ M). Adenosine-2’-phosphate,
adenosine-g’-phosphate,adenosine, inosine-5’.phosphate and ATP were inactive.
ADP had about I per cent of the AMP activity, but it is possiblethat the preparations were not free of myokinase activity. In contrast to the TPN-linked dehydrogenasefrom either animal tissuesor yeast, the DPN-linked enzyme did not react
with oxalosuccinate. The enzyme neither catalyzed decarboxylation nor did it
reduce OSA in the presenceof DPNH. In fact no inhibition of isocitric oxidation was
observed by addition of OSA. It was not possibleto demonstrate reversibility starting with a! ketoglutarate and CO2 under conditions where reductive carboxylation
with TPN-linked enzymes occurred. The enzyme required Mg* or 8InS-t for activity and showed nearly 50 per cent inhibition in the presenceof 0.01 a!t sodium
cyanide or sodium azide.
A. DPN-linked isocitric dehydrogenasefrom animal tissueshas recently been
described (81). It was prepared from acetone dried mitochondria of various tissues
(muscle, heart, kidney and liver). The 40. to so-fold purified enzyme resembledin
many respects the yeast DPN-linked enzyme: metal requirements, lack of reversibility and inhibition by cyanide. However, the animal enzyme did not require addition of adenosine-5’.phosphatefor activity. In view of the other similarities it would
be desirable to know whether the enzyme as well as the DPN preparation were free
of AMP, particularly since somecommercial preparations of DPN contain AMP.
c) ‘PhosphogIuconic’ enzyme. This enzyme has been shown to catalyze the
oxidative decarboxylation of D-6-phosphogluconate to ribulose-s-phosphate (219).
D-6phosphogluconate + TPN+ g D-ribulose-s-phosphate+ CO2 + TPNH + H*.
The properties of this enzyme purified from brewers’ yeast resemblethe ‘malic’
enzyme in several respects. a) No separation of oxidation and decarboxylation activity could be achieved in the course of a procedure which resulted in so-fold purification of the enzyme. b) The hypothetical intermediate, 3-keto phosphogluconate,
could not be isolated or demonstrated. c>As in the ‘malic’ enzyme the over-all process
is reversible and CO2 fixation into D-6-phosphogluconatecould be demonstrated. d)
The enzyme is activated by Mg* or Mntt- and produces a keto compound as end
product of oxidative decarboxylation (cf. 220).
These findings support Horecker’s theory of the oxidation of the p hydroxy
group as the reaction mechanism. However, Uehara has proposed an alternative
mechanism(221). He suggestsan oxidation of phosphogluconateto 2-ketophosphogluconate followed by a cleavage to hydroxypyruvate and triose phosphate. The
latter combineswith decarboxylated hydroxypyruvate on the enzyme to form ribulose-s-phosphate.This alternative hypothesis might be acceptable were it not for
Horecker’s experiments which demonstrated the reductive carboxylation of ribulosej-phosphate to form phosphogluconate. CO2 fixation into hydroxvpyruvate
has
e
not been demonstrated.
Partially purified preparations from rat liver have been studied (222). The enzyme has an alkaline PH optimum (PH 9.0 in glycylglycine) and is slightly stimulated
by someions (AfIg++,Mn++, Ca++ at 10~~M) but strongly inhibited by IO-” M Hgtt.
The Kgnfor 6-PC was found to be 9 X IO-~ M at PH 9.0 and I X row5h5at PH 7.6
as compared to 5 X 10~~M of the yeast enzyme at PH 7.6. The Km value for TPN was
much lessPH sensitive (2.4 X 10~~M at PH 9.0 and 2.8 X IO-~ M at prx 7.6). ,4 number
January
PYRIDIXE
1955
NUCLEOTIDE-LINKED
ENZYMES
33
of SH inhibitors were found to inactivate the enzyme, 2.5 X IO-~ M p-chloromercuribenzoate resulting in 83 per cent inactivation. Cystine, which had no effect on
glucose-6-phosphate dehydrogenase, was found to be highly inhibitory to ‘phosphogluconic’ enzyme. Both nicotinamide and ATP compete with the nucleotide
coenzyme for the enzyme. Of interest is the pronounced difference in enzyme content between the two sexes. Extracts from the female rat tissues contain more than
twice the activity of the ‘phosphogluconic’ enzyme as compared to extracts from
male rat tissues.
Hydroxy acid thiol ester dehydrogenases (fl keto reductases). An enzyme which
catalyzes the reaction
0
1I
RCHOH-CH&-S-CoA
+
DPN+
+
0
0
II
II
R-C-CHH:!-C-S-CoA
+
DPNH
+
H+
has been purmed from liver tissue (223, 224).
The enzyme appearsto react also with the S-acetoacetyl derivative of N-acetyl
thioethanolamine, the CoA analog prepared by Lynen and his collaborators, as well
aswith CoA, sincea 3oo-fold purification of the enzyme from sheepliver extract failed
to separate the activities with the two thiol esters.
From equilibrium studies at PH 7.0 and 7.4 with S-acetoacetyl-N-acetyl thio[acetoacetyl - sR] EDpNH] LH+] = 2 x Io-lO (cf 225)
ethanolamine, K =
.
, wh.1ie
[hydroxybutyryl - SR] [DPN]
for CoA the K value = 6.3 X 10-1~ (225).
This marked difference between the two
values of K may well be an expressionof the influence exerted by the thiol residueon
the equilibrium. However, other factors such as enzyme concentration and effect of
buffers and ionic strength affecting K values have been discussedin connection
with ADH and MDH. A study with the two thiol estersunder,comparableconditions
would be of considerableinterest. It is apparent that the EO for the hydroxyacidketo acid system is considerably raised by combination with a thiol compound,
particularly in the caseof CoA. These shifts in the redox potential may well assume
physiologica. significance in the regulation of cell metabolism. All hydroxv acid
thiol esters of Co-4 from C4 to Cl2 were found to be oxidized by highly p&ified
preparations of the dehydrogenasefrom beef liver mitochondria (224). TPN cannot
substitute for DPN. It has been shown that D-hydroxybutyryl-CoA is the substrate
for the enzyme, the L-isomerbeing inactive (224, 226).
Hydroxyl thiol ester dehydrogenaseswhich react with thiol compounds other
than CoA also exist. Yeast extracts have been found to catalyze the oxidation of
hydroxy thiol estersof glutathione (cf. 93) but neither the products nor the enzymes
have been characterized as yet.
Hemiacetal Dehydrogenases. Enzymes oxidizing hemiacetals.a) Glucose dehydrogenase (glucoseDH). GlucoseDH purified from beef liver (193, 227) catalyzes
the reversible reaction: P-Glucose + DPN+(TPN+) e Gluconolactone + DPNH
[Gluconolactone] [DPNH] [H+] =
(TPNH) + H+. From the K, at PH 6.7 (227) X =
[Glucose] [DPN]
3 X rod. However, Brink (193) reported a K value of 3 X IO-?.
The enzyme acts on glucopyranose and oxidizes it to the lactone by removal of
34
E.RACKER
l~olzcnz
e 35
hydrogens from the hemiacetal. The lactone can be hydrolyzed spontaneously to
gluconic acid, but a lactonase which accelerates the reaction has been described
recently by Brodie (228).
It is conceivable that a phosphorolytic cleavage of the
lactone, analogous to the phosphorolysis of thiol esters, may be used by cells as a
mechanism for trapping the energy of the oxidative step.
SPECIFICITY. Purified glucose dehydrogenaseoxidizes D-xylose as well as giucase, but the pentose is oxidized at one-fourth the rate. The following substances
were unaffected : raffinose, lactose, maltose, CYD-glucoheptose, ,B D-glucoheptose,
D-mannose, D-fructose, D-ribose, D-glyceraldehyde, glycolaldehyde, acetaldehyde
and several phosphorylated hexoses. Galactose and arabinose were very slowly
oxidized compared to glucose.Since crude extracts oxidized these two sugarsat comparatively faster rates (about 20% of that of glucose) the possibility of a separate
dehydrogenasefor thesesugarswas suggested.GlucoseDH preparations of somewhat
higher purity were completely inactive with arabinoseand showedvariable activitk
with different galactose samples (193).
Since galactose obtained commercially is
frequently contaminated with small amounts of glucosethe positive experiments may
have been due to the presenceof glucose.Whether the crude extracts have a galactose
and arabinosedehydrogenaseor a ‘waldenase’type enzyme (which transforms galactose into glucose) coupled to glucose dehydrogenaseremains to be explored. Comparative rates with a and ,&glucopyranose indicated that the latter is the primary
substrate since cu-glucosewas oxidized after a lag period of over I minute and at a
considerably slower rate. Similarly (5xylose appears to be the primary substrate.
The product of the oxidation is suggested to be the &gluconolactone; however
definite evidence on this point is still lacking. The published curves show little
differencesin the rate of utilization of 6- and y-gluconolactone at comparableDPNH
concentrations. These findings are complicated by contamination of the y-lactone by
small amounts of Ei-lactone(227).
INHIBITORS OF GLUCOSE DEHYDROGENASE (193,
229). A number of compounds
structurally related to the pyridine nucleotides have been shown to inhibit the
enzyme. It is concluded from the inhibitory action of nicotinamide, adenine, adenosine, ATP and others, that DPX has several points of attachment to the protein
Pyridoxal and 4-pyridoxic acid are particularly potent inhibitors. Of interest is the
inhibitory action of high concentrations of phosphate in connection with earlier
studies of Harrison discussedbelow. Phosphorylated hexoses,pen.toses,glucose-&
phosphate, fructose-I, 6diphosphate and others are competitive inhibitors of glucose.
Since the binding of glucose-&phosphate is over 10,000
times as tight as that of
glucose, and very small concentrations of glucose-6-phosphateinhibit glucose oxidation, the question of the physiological function of the enzyme has been raised
(193,
229). It has been pointed out previously (153) that the experiments of Stetten
and Stetten, showing a lack of isotopic dilution of urinary gluconate-Clj, have caused
doubt in regard to the contribution made to the gluconate pool by the reaction
catalyzed by glucosedehydrogenase.
A rather interesting observation which does not seem to have been explored
further was made by Harrison over 20 years ago (230). He reported that addition of
xanthine oxidase to a preparation of glucose dehydrogenaseleads to the inactivation of the latter provided a hydrogen acceptor, either oxygen or methylene blue, is
also present. Neither of the two enzyme preparations was pure enough to permit
definite conclusions.The suggestionof the author that an aldehyde group on the
enzyme is functional is worthy of further investigation, particularly in view of his
January
WRIDIKE
1955
NUCLEOTIDE-LINKED
ENZYMES
35
supporting experiments on the reversible inactivation of the enzyme by bisulfite.
Also of interest is the observed protective effect of phosphate against this unique
type of inactivation by xanthine oxidase in view of the reported reactivity of the
enzyme with phosphate and sugar phosphate.
Attempts to detect an absorption shift with DPNH and the enzyme similar
to that describedwith ADH have been unsuccessful(193).
b) Glucose-6-phosphatedehydrogenase (G-6-p DH). G-6-p DH catalyzes the
reversible oxidation of G-6-p to the corresponding lactone (23I, 232). Glucose-6
phosphate + TPN+ e 6-phosphogluconolactone+ TPNH + H+.
A simple procedure yielding a very active preparation of G-6-p DH has been
published (233). Reversibility has been demonstrated with the &lactone of phosphogluconate (232). The gluconolactonase discussedabove catalyzes also the hydrolysis of the phosphogluconolactone (228).
The lL for TPN and for glucose-6phosphateis 1.3 X 10~~ M for the liver enzyme, which is susceptibleto SH inhibitors
such as p-chloromercuribenzoate and iodosobenzoate. Extracts from female rat
tissuescontain about twice the activity of glucose-6-phosphatedehydrogenase as
extracts from male rat tissues. G-6-p DH is considerably more inhibited by phosphate than is glucose DH (222).
G-6-p dehydrogenasefrom E. coli has been shown
to be stimulated by Mg* and Ca++, but inhibited by copper, zinc and other ions
(234)
Thiohemiacetal
dehydrogenases (aEdehyde dehydrogenases). The nucleotide-linked
aldehyde dehydrogenaseswill be provisionally classedin this group with the exception of glyceraldehyde3-phosphate dehydrogenase (TDH) which will be placed in
group II. The reaction catalyzed by the thiohemiacetal dehydrogenasescan be written
as follows:
l
(I)
RICH0 + SHR:! 6 R1 CHOH SR2
(2)
RICHOH-SR:! + DPN’S
(y)
RICSR~
RICSRz + DPNH + H+
II
0
--+ RICOOH + SH R2
+ Et0
II
0
or
0
(3b) RIG-SR2
II
0
+
H3P04
e
RI-C
//
\
+
SHRz
OPOJI2
The evidence for the participation of an SH group is not equally good for each
one of the aldehyde dehydrogenasesdiscussedbelow. Indications for the participation of a thiohemiacetal in aldehyde oxidation can be obtained in those casesin
which one can demonstrate a specific cofactor, e.g. CoA in the bacterial dehydrogenase (130, 235) or glutathione in formaldehyde dehydrogenasefrom yeast (93,
236) or liver (237). When the SH groups of the enzymes participate in the reaction
and no free SH compound is needed, evidence for thiohemiacetal participation is
difficult to obtain. Alternative hypotheses of either aldehyde dehydrogenation to a
ketene, followed by hydrolysis, or oxidation of an aldehyde hydrate must be en-
36
E. RACKER
Vdume
35
tertained. The rather general SH character of most of the nucleotide-linked aldehyde
dehydrogenases
points however to the unified view of thiohemiacetal participation.
a:) Liver aldehyde dehydxogenase. This enzyme although only partially purified from liver has been completely separated from alcohol dehydrogenase and has
no mutase activity (77). It catalyzes the reaction: Acetaldehyde + DPN+ 4
acetic acid + DPNH + Hf. The equilibrium of the reaction is far to the right and
attempts to reverse the reaction have been unsuccessful.
SPECIFICITY. The enzyme reacts with DPN; TPN is inactive. Several aldehydes
are oxidized by the enzyme preparation:-formaldehyde, glycolaldehyde, propionaldehvde, butylaldehyde, isovaleraldehyde, imidazolacetaldehyde (238), salicylaldehyde
and benzaldehyde. Benzaldehyde is oxidized at a very slow rate (239) and inhibits
markedly the oxidation of acetaldehyde (77). This finding and other observations
with inhibitors may be quoted in favor of a single enzyme which activates the aliphatic as well as the aromatic aldehydes. In view of the fact that the enzyme preparations are not pure, the possibility that two separableenzymes are involved should
still be considered.The oxidation of betaine aldehyde by the enzyme preparations
will be discussedlater.
Of considerable interest are the studies of Kendal and Ramanathan (240) on
the formation of estersin a DPN-linked aldehyde oxidation system from horseliver.
In the presenceof formaldehyde and methanol a volatile ester was obtained with
the properties of methylformate. At first the reaction was believed to be due to a
combination of action of ADH and Ald DH giving rise to an oxidation-reduction of
the aldehyde. In such a system the methanol can be visualized as competing with
water for the acylenzyme, giving rise to an alcoholysis instead of a hydrolysis. Since
the enzyme was found to be quite resistant to IAA, the idea of aldehyde dehydrogenaseparticipation was later abandoned. The authors suggest as an alternative
hypothesis that ADH may also catalyze the oxidation of a hemiacetal formed between the aldehyde and the alcohol. Thus ADH could catalyze the complete dismutation. Although a similar possibility has been considered to explain data obtained with crystalline ADH from yeast in the presenceof GSH thiohemiacetals(88)
rigid evidence for oxidation of hemiacetals by ADH is lacking. The experiments
demonstrating a failure of IAA to inhibit the formation of the volatile ester do not
rule out the participation of aldehyde dehydrogenase.The latter enzyme is far less
sensitive to IAA than is TDH and inhibition by IAA is obtained only after considerable time of exposure to the inhibitor. Moreover, little is known about the limiting
factor during ester formation and more direct evidence than the effect of an inhibitor
is needed.
INHIBITORS. Ald DH is an SH enzyme. During dialysis it becomesinactive,
unlesseither ethylene diamine tetraacetate (‘Versene’) or cysteine is added. Once
the enzyme is ‘oxidized’, only partial restoration has been obtained with SH compounds (40). The enzyme is markedly inhibited by tetraethylthiuram disulfide and
by chloralhydrate (241, 242); the latter inhibits competitively with ,the substrate,
the former noncompetitively. The inhibition of the enzyme by these drugs may
have physiological significance, which was discussedelsewhere(88).
The question has been raised of the identity of the liver enzyme and glyceraldehyde-+phosphate dehydrogenase(243) which has been shown also to oxidize nonphosphorylated aldehydes, although at a slow rate. However, there can be little
doubt that liver aldehyde dehydrogenase is not identical with triose phosphate
Janzrcrrq,
I t) j j
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
37
dehydrogenase (TDH) for the following reasons: a) The substrate specificity is
different ; the liver enzyme is inactive with glyceraldehyde-3-phosphate. b) The liver
enzyxne cm be saturated at very low substrate concentrations while TDH is not
saturated by aldehyde concentration 1000 times as high. c> Arsenate or phosphate
doesnot stimulate acetaldehyde oxidation by the liver enzyme,
but doeswith TDPlf.
d) Acetyl phosphate is not reduced by the liver enzyme
but is by TDH. e) The enzymes behave differently during purification and can be separated.
b) Yeast aldehyde dehydrogenases. An enzyme similar to liver Ald DH has
been obtained from yeast (78). The enzyme was found to require SH groups as
well as pot,assiumions for activity, Rubidium or NH*+ substituted for K.+, but Lif,
Na+ and Cs+ inhibited. p-Chloromercuribenzoate inhibited at low concentrations
( IO-4 M), glutathione reactivated. Benzaldehyde was oxidized by the yeast enzyme,
while salicylaldehyde was not. Formaldehyde and other aliphatic aldehydes (including succinic semialdehyde) were oxidized at a rate considerably slower than
acetaldehyde. DPN (Km = 1.3 X IO-* M at PH 8.7 and 0.3 X IO-* M at PH 7.7)
was usedat about the samerate as TPN. The purified enzyme contained only negligible amounts of pantothenic acid or lipoic acid.
A TPN specific enzyme has been isolated from bakers’ yeast (244). It differs
from the above yeast enzyme in its lack of a K+ requirement. Instead, it is stimulated
by divalent cations (Ca*, Mgft, Ba* or Mn.+f). Acetaldehyde, glycolaldehydepropiona1dehyd.eand formaldehyde were shown to act assubstrates.Km for acetaldehyde = 3.5 x Io-5 III, for glycolaldehyde = 2.2 X 10~~ M and for TPN = 1.4 X
IO---~ M. No data on the susceptibility of the enzyme to SH inhibitors are recorded.
c) CoA-linked bacterial dehydrogenases. Extracts from CL Kluyveri (235) and
from E. col-i (130) contain an enzyme which catalyzes the reaction: acetaldehyde +
CoA + DPN+ ; acetyl CoA + DPNH + H+. Crude CL kluyveri extracts react
with DPN and TPN, but with partially purified enzyme only DPN is active. Glycolaldehyde, propionaldehyde and butyraldehyde were oxidized at about one-third the
rate of acetaldehyde. Formaldehyde, chloral and benzaldehyde were negative. No
oxidation occurred in the absenceof CoA SH, which was required in stoichiometric
[acetyl CoA] [DPNH] [H+]
amounts to yield acetyl CoA as the end product. K =
[acetaldehyde] [DPN]
=
I.2 x
10 --d.From the equilibrium constant and the AF of the DPN-linked oxidation
of acetaldehyde to acetic acid a AF for the hydrolysis of acetyl CoA of 13,040calories
was calculated, which appears to be in good agreement with other data (245), but
using a slightly different set of values for the DPN potential and the oxidation of
acetaldehyde to acetic acid, a value of about - 8000 calorieswasobtained. This latter
figure
is in closer agreement with the AF of hydrolysis for other thiol esters (246).
d) Betaine aldehyde dehydrogenase. A stimulation of betaine aldehyde oxidation by DPN was observedby Klein and Handler (161). As mentioned above betaine
aIdehyde is oxidized by purified preparations of Ald DH from liver, a reaction which
is also inhibited by tetraethylthiuram disulfide (241). Recently another DPNlinked enzyme from rat liver, which is apparently specific for betaine aldehyde and
is an SH enzyme has been obtained by Rothschild and Barron (165). The partially
purified enzyme requires cysteine or GSH for activity. Acetaldehyde and glyceraldehyde (IO-~ M) inhibit betaine oxidation about 50 per cent. Formation of a thiohemiacet.aI as an intermediate has been suggested(165).
FORMALDEHUDE
DEHYDROGENASE.
A DPN-linked formaldehyde oxidizing en-
E. RACKER
38
zyme (93) has been partially purified from yeast (236) and from liver (237). Both
enzymes require glutathione6 as cofactor and are DPN specific. The liver and yeast
enzyme has been found to be inactive with acetaldehyde and various other aldehydes
but the yeast enzyme oxidizes methyl glyoxal as well as formaldehyde (236). It is
possible however that i-he activity with methyl glyoxal is due to a separate‘enzyme.
BENZALDEHYDE
DEIIYDROGENASE.
Extracts from Pseudomonas jluoresce~~s grown
on n-]L-mandelatecontain 2 separable enzymes oxidizing benzaldehyde (247). One
is a DPN-, the other a TPN-linked dehydrogenase.Since even crude extracts contain
little dehydrogenaseactivity for aldehydes other than benzaldehyde, the enzymes
are consideredto be rather specific benzaldehyde dehydrogenasesinduced by mandelate or its meta.bolicproduct,s.
Formic Dehydrogenase. This enzyme catalyzes the reaction, HCOOH + DlW+
* CO2 + DPNH + H+. DPN-linked formic dehydrogenasehas been purified from
pea seeds(248-250). The reaction is irreversible for all practical purposes,sinceinsignificant amounts of C1402were incorporated into formic acid (249). Formic dehydrogenaseis one of the few nucleotide-linked dehydrogenasesreported to have a high
sensitivity to cyanide. At IO-~ M concent-ration cyanide (248, 249), azide and pchloromercuribenzoate (250)
inhibit the activity nearly completely while Fe+++,
Cu++ and iodosoben’zoateinhibit strongly. IAA has no effect. The inhibition due to
cyanide and azide is reversed by dialysis. These interesting properties of formic
dehydrogenaseare not unlike those of other enzymes which require SH groups as
well as a metal for full act,ivation.
Group II
The enzymes to be cliscussedin this group are consideredto catalyze a cleavage
of an S-R or an S-S bond by an aldehyde or an a-ketoacid. The processresults
in the formation of a thiol ester and a reduced cofactor. Since this type of aldehyde
and keto acid oxidation has received considerableattention in the past few years
(4, 13, 93), the discussionhere will be limited to an analysis of more recent developments and to aspectswhich have received lessattention in other reviews.
Glycerafdehyde-3-Phosphate Dehydrogenase (Triose Phosphate Dehydrogenase = TDH). TDH has been obtained as crystalline protein from yeast ancl
from rabbit muscle (cf. 72). It catalyzes the over-all reaction:
CHPOP03H2CHOHCH0
+
DPN+
+
HP04=
e
0
CHPOPO;1H2CHOHC
/
\
+
DPNH
+
H’
OPO.7=
The enzyme requires free sulfhydryl groups for activity and unlessprecautions
are taken during the isolation procedure, particularly from rabbit muscle,the enzyme
is obtained in the ‘oxidized’ form, i.e. it requires addition of an SH compound for
maximal activity. It has been found (45) that if the isolation of the muscleenzyme
is carried out in the presenceof a metal binder, e.g. KCN or ethylene diamine tetraacetate (EDTA), addition of an SH compound is not required. The enzyme is ‘fully
6 Miss Zelda Budenstein
at Yale University
has recently synthesized
what
glutathione.
The synthetic preparation
when added to the purified yeast enzyme
of DPNH.
appears
resulted
to be fox-my1
in oxidation
hnuary
1955
PYKIDINE
NC’CLEOTIDE-LINKED
ENZYMES
39
reduced.’ It should be mentioned here that for the st,udy of the mechanism of action
it has been important to deal with ‘fully reduced’ enzyme and several misinterpretations are in the literature because of the use of ‘oxidized enzyme.’ On the other
hand it is difficult to define the fully reduced enzyme, since reactivation by SH compounds does not necessarily give full reactivation and varies with the different SH
compounds used. From studies to be discussed below it may be concluded that
fully reduced enzyme can be obtained by increasing the concentration of EDTA IOfold over that originally recommended (45) or by treatment of partially oxidized
enzyme with BAL (128).
An analysis of the amino acid content and of the end groups of the muscle and
the yeast enzyme (70) revealed a high valine and low glutamic acid content in both
proteins. Amino end group analysis yielded two valine end groups. No glutamic acid
end group was obtained. The striking similarity between the two proteins has been
emphasized (72). In fact it is felt that someof the quoted dissimilarities, with the
exception. of the immunological differences, may be more apparent than real and
due to differences in preparative methods and test conditions. For instance the
striking difference in stability of the two enzymes in the absenceof DPN disappears
when the muscle enzyme is fully reduced and metal contamination is avoided. Although undoubtedly minor differences exist, the two proteins obtained from so
vastly different sourcesshow a remarkable unity of properties.
TDH hasbeen shown to bind DPN (83). Recent experiments with fully reduced
enzyme have shown that up to 3 molesof DPN can be obtained from each mole of
several times recrystallized muscle TDH (so).
TDH contains also at least 2 moles of firmly bound gluta thione per mole of
enzyme (I 28). The mode of binding of GSH has not been established. GSH is not
removed by treatment with charcoal, which removes bound DPN. It should be
stressedthat free glutathione is adsorbed quantitatively on charcoal under these
conditions. GSH is releasedfrom the enzyme after mild digestion with trypsin. This
fact cannot be taken as evidence for a peptide or ester bonding of GSH since alteration in protein configuration may alter the binding properties of the residue. From
amino end group analysis (70) it appearsthat GSH doesnot form a free amino end
group. Provided that the pretreatment for end group analysis has not lead to a releaseof GSH which would be missedanalytically, one may suggestthat GSH is in
a peptide chain, or is bound as a cyclic peptide.
The binding of DPN to SH groups of the enzyme was first suggestedby Rapkine (47) who observed protection of the enzyme by DPN against some oxidizing
agents. More direct evidence was obtained by spectrophotometric studies which
demonstrated an absorption at 360 rnp due to the DPN-enzyme complex and its
disappearanceafter treatment with SH inhibitors (45). Quantitative studies on the
effect of IAA on the releaseof GSH by proteolytic digestion strongly suggestthat
bound GSH is the binding site for DPN (128).
These experiments will be discussed
later.
SpeciJi~i~. Glyceraldehyde-3-phosphate is the most rapidly utilized substrate
ulyceraldehyde, butyraldehyde, glycolfor TDH. Acetaldehyde, propionaldehyde, b
aldehyde, succinic semialdehydeand methyl glyoxal have been shown to be oxidized
at considerably slower rates (251).
Formation of acyl phosphate with most of these
al&hy&s has been demonstrated by hydroxamic acid formation. No evidence for
the formation of the acyl phosphate has as yet been published in the caseof glycera&+ydc. Reversibility of the over-all reaction is readily demonstrated with I ,3
diphosphoglyceric acid or acetyl phosphate and DPNH. The depenelcnre on phosphate or arsenate is a function of the substrate used. Without phosphate, 1.
qlvceralde4
hyde-s-phosphate
is hardly oxidized at all by TDH, while with some mu~le ‘I’DH
preparations, glyceraldehyde is oxidized at the same rate in the presence ;JS in the
absence of phosphate or arsenate. With acetaldehyde, a pronounced arsenate effect
can be readily observed. This variation in phosphate dependence of the oxidation
of different substrates is probably a reflection of the stability of the various acyl
enzymes, e.g. glyceryl-enzyme being more labile than acetyl-enzyme. It is of interest
to note that a similar labilization due to the presence of a hydroxyl group has been
observed with thiol esters of GSH, e.g. glycolyl glutathione being more M~ile t ban
acetyl glutathione.
It has been known for many years that arsenate can replace phospl~te in the
reaction catalyzed by TDH. It has been proposed that arsenate acts similarly to
phospha.te by forming a I-arseno+phosphoglycerate,
which is believed to be very
labile and to be hydrolyzed spontaneously into free phosphoglyceric acid and arsenate. Alternatively,
the possibility may be suggested that the 3.-arse~lo-~~-phosphoglycerate never occurs in solution but that arsenate and phosphoglycerate are
separately released by the enzyme. This explanation is in line with the ability of the
enzyme to hydrolyze acyl phosphates, a reaction which will be discussed later.
Mechanism uj action. From the studies of kinetics and transfer reactions of
acyl and phosphate groups which have been reviewed elsewhere(93) the fr)llowing
z-step reaction sequencehas been shown to occur:
reaction
a)
0
/
RC
\ ‘\
‘H
+
DPN+
+
Enzyme-SH
r”
0
//
R C-S
Enzyme
+
DPN 1-I -+- I-l+
0
reaction
b)
//
RG-S
Enzyme
+
phosphate e
0
RC
/
+
3:nzynle- 231
\
Sum:
‘OPOsHz
______--_-l___ -------------0
/
RCC
+ DPN+ + phosphate r‘
‘\
“H
--__...
-..
0
RC
/
\
+
DPXII
OPO~H’L
The evidence for this reaction sequencemay be analyzed as follows:
$-
11’
January
1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
41
I) Reaction (b), the phosphorolytic step, can be functionally separated from
oxidation-reduction since arsenolysis as well as P32 exchange has been shown to
occur in the absenceof added DPNH. The evidence is not entirely conclusive since
bound DPN is required for these reactions and a shuttling mechanism involving
oxidation-reduction of bound DPN could not be entirely ruled out. However, IAAtreated enzyme, which did not catalyze oxidation-reduction even in the presenceof
GSH, did catalyze arsenolysisif GSH was added.
z) The enzyme catalyzes an acyl transfer to either GSH or CoA. This reaction
can be carried out from either the aldehyde or the acyl phosphate and does not
require inorganic phosphate; in fact the latter inhibits the reaction.
-7) The occurrence of oxidation without phosphorylation, e.g. with glyceraldehyde as substrate, has been frequently cited as evidence for the formation of acyl
enzyme. This cannot be consideredsatisfactory evidence, since it is obvious that the
dehydrogenation of an aldehyde hydrate or the formation of a ketene could be an
alternative explanation for this observation; in the case of the aldehyde dehydrogenasesfrom liver and yeast which are not stimulated at all by phosphate attempts
to detnonstrate the formation of acyl enzyme have so far failed.
4) Perhaps the most cogent evidence has come from the direct demonstration
of acyl enzyme formation. Using enzyme as stoichiometric reagent with acetyl phosphate assubstrate, up to I .8 equivalents of acyl groupshave beenshown to be formed
on t,he enzyme. Two methods have been used to stabilize the enzyme-substrate
compound, both consisting of a processof enzyme inactivation. The enzyme was
either boiled at PH 4.5, at which acetyl phosphate is destroyed; or the hydrolysis
was prevented by removing DPN from the enzyme. Since DPN is required for the
hydrolysis or phosphorolysisof the acyl enzyme, stable preparations of acyl enzyme
can thus be obtained.7Acyl enzyme has also been obtained with the natural substrate
glyceraldehyde-g-phosphate.
The TDH-DPN complex has been shown to exhibit an absorption with a flat
maximum at. about 360 rnp, which was diminished by addition of SH inhibitors
such as IAA or p-chloromercuribenzoate, by oxidation of the SH with oxidizing
agents such as Hz02 or iodine and finally and most significantly by the addition of
the substratesacetyl phosphate or I ,3 diphosphoglycerate. From these experiments
it was proposed that a) the DPN-S-enzyme complex participates in the catalysis
and b) a cleavage of S-C bond of the complex by the aldehyde takes place (aldehydolysis) resulting in the formation of acyl enzyme and reduced DPN. This process
of aldehydolysis was proposed in preference to the previously suggestedmechanism
of thiohemiacetal formation. However, other investigators have favored the thiohemiacetal as the primary intermediate becauseof the pronounced protective effect
of 3-phosphoglyceraldehyde against IAA inhibition (127, 129). Since the phosphorylated substrate is an effective protector even in the absenceof DPN, the formation of a thiohemiacetal as the primary step was suggested,and a correlation between
the protective ability and effectivenessas substrate was pointed out (129). A more
detailed study of the protection mechanism revealed that such a correlation does
not exist,. Substanceswere found, like 3-phosphoglycerate, which neither serve as
substrate nor are capable of thiohemiacetal formation, but which effectively protected TDH agai,nst IAA (128).
It is clear from these experiments and from the
7 Crystsllitle
preparations
of acyl enzyme
(free of DPN)
have been recently
obtained
by Dr.
I‘. Krimsky
in our laboratory.
These preparations
gave a positive hydroxylamine
reaction even after
boiling at plr ‘4.5 (which destroyed
any residual acetyl phosphate).
When DPNH
was added to large
amour&
of acy1 enzyme,
reoxidation
of the nucleotide
and formation
of acetaldehyde
took place.
42
E. RACKER
Vohbme 35
high affinity of glyceraldehyde-3-phosphate that the substrate interacts with the
enzyme at more than one site and eliminates the objections raisedby Segal and Boyer
(I 2 7) against the aldehydolysis mechanism.
When prior to the addition of DPN, acetaldehyde was added to the enzyme to
permit the primary formation of the thiohemiacetal, a marked inhibition (over 90 %)
of the reaction rate was observed (so). The observation is dficult to reconcile with
the theory of hemiacetal formation and favors the participation of the DPN-Scomplex in the reaction.
The evidence for the participation of bound GSH in the reaction mechanism
rests on the following:
1) Glutathione is releasedfrom the enzyme by proteolytic digestion. Addition
of 3 equivalents of IAA, which are required to abolish the activity and the 360 rnp
absorption complex, completely blocks the releaseof free GSH by tryptic digestion
of TDH.
2) N-ethyl maleimide in excessalso inhibits the enzyme and the tryptic release
of GSH. In the presenceof the substrate, acetyl phosphate, the activity as well as
the releaseof free GSH is protected.
3) Digestion of acyl enzyme with trypsin leadsto the releaseof a thiol ester with
the properties of acetyl glutathione (128).
The crystalline enzyme has been shown to catalyze an oxidation-reduction
step and a phosphorolvsis. In addition the following activities are catalyzed by
crystalline TDH:
a) Phosphotransacetylase.In the presence of acetyl phosphate and GSH or
CoA the formation of acetyl glutathione or acetyl CoA has been shown (45, 59).
However, the reaction proceeds at a slow rate and is very likely dependent on a
nonenzymatic acyl group transfer similar to that observed by Stadtman (I IO) and
by Wieland (IO). Acyl enzyme + RSH e acyl-SR + SH enzyme.
b) Thiol esterase.Thiol estersare slowly hydrolyzed by TDH. This reaction too,
is probably dependent on the chemical acyl transfer and secondary hydrolysis of
acyl enzyme in the absenceof acyl acceptor.
c) TDH has been shown to catalyze the reaction: DPNH -+ DPNX (252)
which can be followed by the lossof absorption at 340 rnp. The structure of DPNX
is unknown but supposedly identical with the compound obtained when DPNH
is exposed to acid solution. DPNX is inactive as oxidation-reduction catalyst, but
can be partially converted into DPN by crude yeast extract in the presenceof
AT-P (253).
d) Acyl phosphatase. Acetyl phosphate has been shown to be hydrolyzed by
oxidized enzyme (59). Similar observations have been made with ‘fully reduced’
enzyme, demonstrating considerable instability of acyl enzyme in the presenceof
DPN. Harting’s observation of an accelerated phosphatasereaction with oxidized
enzyme was confirmed. IAA-treated enzyme was a.lsofound to have a higher phosphatase activity than reduced TDH. Both GSH and GSSG inhibited the hydrolysis
of acetyl phosphate by the enzyme (so).
From these observations one might speculate about the significance of other
‘phosphatase’activities found so frequently in cells damaged by enzymologists or
by histochemists. Could they (the phosphatases)be artifacts? Could an important
function such as that of a group transfer have ‘degenera.ted’into a hydrolytic process?It is of interest in this connection that some of the phosphataseshave been
reported to be inhibited by SH compounds or activat,ed by iodoacetate like the
phosphataseactivity of TDH (cf. 254).
January
PYRIDINE
I955
NUCLEOTIDE-LINKED
ENZYMES
43
It is apparent from the above discussion that TDH looks more like a Medusa
head than a simple double-headed enzyme. In summary one can outline the activity
of this enzyme as follows: Under ‘normal’ conditions with fully reduced enzyme,
the first step in the forward and back reaction is the formation of acyl enzyme. If the
enzyme is deprived of DPN, acyl enzyme is stabilized. In the back reaction with
acetyl phosphate, the acyl enzyme is reduced by DPNH and acetaldehyde is formed.
In the forward reaction after the oxidative step has occurred the acyl group is transferred to another site on the enzyme and in the presence of phosphate or arsenate, it
dissociates as acet.yl phosphate or as acetate and arsenate. This other site, the existence of which has been postulated because of the occurrence of arsenolysis in IAAtreated enzyme, is probably identical with the site responsible for the phosphatase
activity. Both activities are dependent on the presence of DPN. If the enzyme is
oxidized or blocked by IAA, the hydrolysis takes preference over transfer since no
acyl acceptor group is present. The inhibitor effect of GSH and GSSG on the hydrolysis of acetyl phosphate by TDH remains to be explained. An interaction between these
inhibitory compounds and the enzyme is indicated by these experiments and is also
suggested by the stimulation of arsenolysis by GSH using IAA-treated enzyme.
Pyruvic Dehydrogenase System. The enzymes which participate in the oxidative decarboxylation of pyruvic acid to acetyl CoA have been proposed to catalyze
the following series of reaction:
/
0
[ 1
0
(I)
CH3-C
\
ThPP,
--
CHs-C
*
CH3C
\
COOH
S
\
0
(2)
Mg++
//
\
/
+
H
S
/
0
II
(7 I?;(-C-S
R
+
CoA
\
-+
/
HS
DPN+
-__-------
R
/
HS
\
(4)
H
\
H-S
f,;)
+
HS
HS
\
HS
/
co2
0
II
CH3-C-S
-+
R
+
R
0
II
+
CH3--C-SCoA
/
s
R
=
\
S
R
+
DPNH
+
H+
/
-
-
ThPP,
Mg++
lipox
acid
r-4 Sum: pyruvate + DPN+ + CoA --yr--+
Acetyl CoA + CO2 + DPNH + II+
According to Gunsalus (255)
lipoic acid and ThPP function separately and
the enzymes which catalyze reactions 3 and 4 have been fractionated away from the
44
E. RACKER
Volzme
35
enzyme or enzymes catalyzing the oxidative decarboxylation. According to Reed
(x3) lipoic acid in amide linkage with ThPP is the functional coenzyme, called
lipothiamide. It appears that both lipoic acid and lipothiamide may function as
acyl and electron transport agents in react&s r-4. Xf lipothiamide can be shown
to work with greater efficiency, we may be dealing here with another example of a
polyfunctional catalyst -a double-headedcoenzyme. As in the case of the doubleheaded ‘malic’ enzyme, a similar efficiency may be operative with lipothiamide, permitting CO2 fixation into an CYketo acid during photosynthesis, an attractive theory
suggestedby Reed (13). However, the available data on CO2 fixation into cyketo
acids, even in crude systems which should contain lipothiamide, are not very encouraging. An alternative hypothesis has been proposedby Calvin (256) postulating
the direct fixation of CO2 into pentose phosphate. Suggestive evidence in favor of
pentose phosphate participation in CO2 fixation is forthcoming (257, 258).
Theories concerning the mechanismof pyruvate decarboxylation and formation
of ‘active aldehyde’ have been published (13, 255).
Reaction 3 represents a thiol
transacetylase reaction. Enzymes from 23. coli (259,
CL ktuyveri and pigeon liver
(259) have been shown to catalyze this type of transfer reaction with reduced lipoic
acid. Several other SH compounds such as 2-mercaptoethanol and H& are also
active. The enzyme from liver has been separated into two fractions, one reacting
preferentially with lipoic acid or 2-mercaptoethanol, the other reacting with I~$.
Reaction 4 is catalyzed by lipoic acid dehydrogenaseand will be discussedlater.
c11Ketoglutaric
Dehydrogenase.
The oxidation of CYketoglutarate to succinyl
CoA is believed to be analogousto pyruvate oxidation (4, 255). However, no evidence
for the formation of succinyl lipoic acid has as yet been obtained.
New advances have been made in our knowledge of the stepsfollowing succinyl
CoA formation. For the enzymes obtained from animal tissuesthe reactions have
been written as (260) :
(a,) succinyl CoA + Pi + GDP + succinate + CoA + GTP
(b) GTP + ADP e GDP + ATP
In the reactions catalyzed by a highly purified enzyme from spinach, evidence
for a phosphate acceptor other than ADP has not been obtained (26oa). Phosphate
transfers between nucleotides of the type of reacfiofz b have become increasingly
recognized in recent years (261, 262).
The mechanismof the actual incorporation of inorganic phosphate in the course
of reactiorz a into the nucleotide is still obscure. The fact that at least in the animal
enzyme system the final phosphate acceptor is GDP may complicate the interpretation of the isotope exchange data. The presenceof a second nucleotide and the
enzyme equilibrating it with ATP may account for some of the findings with P32labeled ADP. The purified spinach enzyme catalyzes the incorporation of V4labeled succinate into succinyl CoA and I332-labeledADP into ATP. However, for
the incorporation of inorganic P3:! into ATP the complete system containing succinate and CoA is required. Succinyl phosphate has been ruled out as intermediate
and accumulation of a free CoA phosphate has also been made unlikely (263).
With the availability of GDP and highly purified enzyme, isotope data with Wlabeled GDP may vield information in regard to the sequenceof interactions.
P-Aspartyl
Se*mialdehyde
Dehydrogenase.
Black and Wright (264) recxently
discovered an enzyme in extracts from bakers’ yeast which catdyzes
the reaction,
Jawary zg55
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
45
P-aspartyl semialdehyde+ H POd- + TPN+ L p-aspartyl phosphate + TPNH +
H+.
The enzyme appears to bear a number of similarities to triose phosphate dehydrogenase. It oxidizes reversibly an aldehyde to an acyl phosphate, and catalyzes
the cleavage of P-aspartyl phosphate in the presenceof arsenate. The oxidation-reduction as well as the arsenolysisare inhibited by iodoacetate.
Grotip III
AmiaQ Acid Dehydrogenases. The enzymes in this group catalyze a dehydrogenation processwhich results in the formation of a double bond between two carbon
atoms or two nitrogen atoms or a carbon and nitrogen atom. Most of the enzymes
in this group have not been studied very extensively and will not be discussedin
very great detail.
Am&o acid dehydrogenases.
The only well-establishedoxidative deamination of
an amino acid by a pyridine nucleotide-linked enzyme is catalyzed by L-glutamic
dehydrogenase.
There is good evidence for the participation of DPN in a number of oxidationreductions of other amino acids particularly in some anaerobic microorganisms
(75, 265). But wheth er these reactions are actually catalyzed by specific dehydrogenaseshasnot beendemonstrated. It was shownthat in extracts from CL sporogenes,
DPN is reduced in the presenceof alanine, leucine, valine and other amino acids.
However, the rate of DPN reduction with alanine, the most active of the group,
is still about one-tenth as rapid as with L-glutamic acid. It is therefore apparent
that the mechanismproposedby Braunstein (266) may be operative in this system:
RNHs + a! ketoglutarate +
R-COCOOH
+ glutamate (transaminase)
glutamate + DPN+
e cy ketoglutarate + NH3 + DPNH + H+ (glutamic DH)
Sum: RNHZ + DPW g1utamate)
RCOCOOH + NH3 + DPNH + H+
As can be seenfrom the experiments of Nisman (79, there are a number of
features which actually favor this alternative explanation. KCN and hydroxylamine,
which are inhibitors of transaminase,also inhibit ‘alanine dehydrogenase.’Glutamic
acid, which acts catalytically, can be shown to arise by proteolysis in well dialyzed
bacterial preparations. Although it may be pointed out that someother nucleotidelinked dehydrogenasesare inhibited by KCN (formic DH and isocitric DH) and
by hydroxylamine (ADH and glutamic DH), the slow oxidation of the amino acids
by CL sporogenes
extracts and their inhibition by aldehyde binders make it advisable
to postpone acceptance of these specific dehydrogenasesuntil they can be fractioned free of glutamic dehydrogenaseor exceed it in activity.
The problem of the hydrogen acceptor amino acids in these anaerobic bacteria
is of considerableimportance. For example, glycine or proline can serve as acceptors
in the DPN-linked system. In neither casehowever is evidence for a direct transfer
of hydrogen from DPKH to the amino acid available.
G&am,ic dehydrogenase
(gZ&amicDH). Glutamic DH hasbeen crystallized from
E. RACKER
46
1~~oltcm.e35
beef liver (115, 194). It catalyzes the reaction, Glutamate
e cy ketoglutarate + NH4+ + DPNH(TPNH)
+ Hf
H20
K
=
[ketoglut~r~~el
w34+1
[glutamate]
WPNHI
[DPN+]
m+1
=
2 x
$- DPN+(TPN+)
Io-15
$-
&I (1,34)
[H20]
Despite the very low equilibrium constant, which is of the same order as that
of the glycolic-glyoxylic
acid system, the forward reaction is much more readily
demonstrated with glutamate than with glycolate. This is understandable in view
of the participation of water in the former reaction.
a) Specificity. The enzyme reacts with DPN, desamino DPN or TPN as coenzymes. The rate is most rapid with DPN and about one-third as fast with TPN.
The rate with desamino-DPN
is about 60 per cent that of DPN. The only amino
acid which is oxidized by the enzyme is L-glutamic acid. Every derivative of glutamic
acid, which has been tested including glutamine and N-acetyl glutamic acid, was
found to be inactive. D-glutamate was not oxidized and in fact. at equal molar concentration it inhibited the oxidation of I.-glutamate by so per cent. Km values for
DPN and DPNH are about the same (IO-* M), for L-glutamate 1.1 X IO--~ M, for
Q! ketoglutarate 0.7 X IO-~ M, for ammonia 5.6 X 10~~ M. Somewhat lower values
for K, of DPN, DPNH and a ketoglutarate were reported by Strecker (1x5).
b) Inhibitors. In addition to D-glutamate, glutamine and aspartic acid were
also found to be inhibitory. Several metals were inhibitory at very low concentrations (Ag+ at 10~~ M). The inhibition by silver ions was not reversed by GSH, while
that by p-chloromercuribenzoate
(IO-~ M) was. At high concentrations anions were
inhibitory (194).
c) Mechanism of action. It has been proposed that the reversible oxidation of
glutamic acid goes through the imino-glutaric acid as an intermediate (267). This
compound was supposed to decompose and to be formed spontaneously. There has
been little evidence for this concept and the kinetic evidence presented by Strecker
( IIS) points against the chemical formation of this compound in the course of the
reaction. The kinetic approach which was used consists in a study of the effect of
one reactant on the apparent Michaelis constant of the second reactant. Essentially
the same method has been used by Bticher and Garbade for the study of a possible
interaction between reactants (66). This fruitful approach has not been very widely
used. Some objections have been raised to conclusions drawn from this type of kinetic
data (73). These studies make it unlikely that spontaneously formed imino-glutaric
acid is the intermediate in the reaction. An alternative mechanism, consisting of an
unsaturation between carbon 2 and 3, followed by enzymatic deamination, was
essentially suggested many years ago (268, 269) :
I
1
CHNH2
6OOH
I
II
I
CH:!
CH
CH2
s
CNH2
t’OOH
C
HtO
II
NH3+C-OH
I
COOH
This mechanism could lead to the formation
i
*
6-O
I
COOH
of an enol rather th.an the keto
Junzlary 1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
47
form of a-ketoglutarate and should result in a labilization of the hydrogen at carbon 3. Experiments with deuterium may help to exclude this possibility.
No interaction between DPNH and glutamic DH could be detected spectraphotometrically and no shift in equilibrium at higher enzyme concentrations similar
to that with ADH was observed (194).
Dihydroorotic
dehydrogenase and others. From a microorganism selected in a
medium enriched with erotic acid an enzyme has been obtained (116) which catalyzes the reactions, Dihydroorotic acid + DPN+ s erotic acid + DPNH + H-t.
The enzyme appears to be specific for erotic acid (Km = 1.1 X IO--~ a6>.No activity
as measuredby DPNH oxidation was found with uracil, cytosine, s-methyl cytosine
or thymine nor did any of these compoundsinhibit the enzyme. An interesting result
was observed with Mg ++ which had no effect on the initial rate of the reaction, but
stimulated when the reaction had proceeded for 20 minutes. Cysteine doubled the
activity of the partially purified enzyme.
There are several other examples of enzymes which may belong in this category. The TPNH-linked enzymatic cleavage of the hepatic carcinogen +dimethylaminoazobenzeneto NIN-dimethyl-p-phenylenediamine and aniline, representing a
reduction of RI - N=N - Rz to RlNHz + RzNH2 (270), the reduction of dihydrofolic to tetrahydrofolic acid by DPNH (I 17), and the saturation of some
unsaturated steroids by TPNH (271) belong in this group. Also the DPN-stimulated
oxidation of cholesterol may have an unsaturated C=C bond as an intermediate
(2 72). The conversion of L-phenylalanine to tyrosine by a rat liver preparation
(273) and the degradation of 2-C l4 lysine to acetate, butyrate and ammonia by an
enzyme from a Clostridium (274) are reactions requiring DPN. It should be made
clear, however, that in many of these casesthe oxidation of reduced nucleotide may
be mediated by a flavin nucleotide and further study of the enzymes will be necessary to decidewhether a direct transfer of hydrogen from the reducedpyridine nucleotide takes place or is mediated by a flavoprotein or even by a cytochrome enzyme.
The latter is implicated in the slow oxidation of DPNH in the presenceof fumarate
and heart muscle preparations (275).
Group
IV
This group consistsof enzymes which catalyze hydrogen transfers with SH and
SS compounds. As mentioned previously, very marked variations exist among the
EL values of various thiol systems, ranging from the verv negative lipothiamide
(-0.42
cf. ref. 13) to the positive GSH (+0.04).
It is apparent that the position
of the equilibrium of a DPN- or TPN-linked system (EL = - 0.32) will depend on
t.he particular thiol compound involved. The finding of a cystine reductase in yeast
(276) is of interest sinceit may permit evaluation of the potentiometric data for the
redox potential of cysteine (-0.14)
by an independent method.
It has been observed in our laboratory that somewell-dialyzed protein fractions
obtained from various tissues (e.g. liver, yeast) catalyze the reduction of DPN or
TPN in the absenceof any added substrate. This activity, which has been ascribed
in laboratory jargon to ‘nothing dehydrogenase,’is very likely associatedwith SH
groups in the fractionated protein and may be related to earlier observations of
Hopkins and his collaborators on the reducing capacity of well washed‘tissueresidue’
(277). The protein fractions from liver and yeast which catalyze DPN reduction
E. RACKER
48
V&me
35
give a strong nitroprusside test and may contain SH groups with a low enough potential to reduce the nucleotide.
Glutathione Reductase (GSSG Reductase). The reaction catalyzed by this
enzyme may be written as follows: GSSG + TPNH + H+ --) 2 GSH + TPN+.
The enzyme has been demonstrated in plants, animal tissuesand microorganisms.
For all practical purposesthe reaction catalyzed by the enzyme is irreversible, which
is understandable from the large potential difference of abdut 0.35 volts,
Spec@city. The enzyme from wheat germ has been found to be specific for
glutathione and not to react with cystine, homocystine, y glutamyl cystine or asparthione.
T:he enzyme has been repeatedly stated to have an absolute specificity in regard
to TPINH (54, 55). Enzyme preparations of GSSG reductase from yeast and beef
liver (57) were found to react quite readily with DPNH provided the test was carried out in phosphate buffer, inhibiting anions were avoided, and rather high concentrations of nucleotide were used. Because of these factors and the considerably
slower rate with DPNH, the enzyme can nevertheless be used for the specific determination of TPNH under appropriate conditions. The enzyme from yeast, which
has been purified about 3oo-fold, did not react with other S-S compoundstested
(cystine, homocystine, cystinyl diglycine, lipoic acid and oxidized CoA). The mechanism of phosphate activation of the reaction with DPNH is of particular interest.
It was shown that the stimulation was not due to enzyme stabilization, but attempts
to demonstrate a phosphorvlated intermediate with inorganic phosphate labeled
with P32have thus far failed.. The reduction of S-S compoundshas received considerableattention in connection with speculationson photosynthesis and the formation of a thiyl free radical was suggested(256). The stimulation of GSSG reductase
by phosphate was not influenced by light.
Cystine Reductase. An enzyme from yeast was found (276) to catalyze the
reaction cystine + DPNH + H+ + 2 cysteine + DPNf.
Little information is available on this enzyme, but from the Ei value for cysteine
( -o.L+)
it may be predicted that it should be possibleto reduce DPN by high concentrations of cysteine in the presenceof cystine reductase.
Lipoic Dehydrogenase. The reaction for the enzyme or enzymes which catalyze
the reduction of DPK by lipoic acid or by lipothiamide has been written as follows:
SH
R
/
\
S
+
SH
DPNC
:R
/
\I
+
DPNH
+
H+
S
The equilibrium with lipothiamide is estimated to be far to the right (Ei =
ref. 13).
The enzyme has been purified from IZ. c02i (255).
Hydrogenases and the ‘Hill Reaction.’ The listing of these enzymes in this
group is arbitrary and may have to be revised on further analysis.
CL khyveri (2 78) and a hydrogenomonas(279) contain a hydrogenase which
catalyzes the reduction of DPN by molecular hydrogen. The enzymes are activated
by iron, cobalt and som.eother divalent metals. No direct evidence for the participa-0.42:,
Jarmary
1955
PYRIDINE
NUCLEOTIDE-LINKED
ENZYMES
49
tion of SH groups in this reaction is available; however, stimulatory effects by SH
compounds have been observed (279).
Calvin (256) has reported a stimulation of the photochemical cleavage of water
by lipoic acid, when the latter is added to the intact cells. The relation of the Hill
reaction to pyridine nucleotide-linked systems has been elaborated elsewhere (2 80).
Groz@ V9 lbcleotide Transhydrogenases
The enzymes in this group catalyze the reaction: DPNH(TPNH)
+ II+ +
pyridine (flavin) nucleotide e DPN+ + reduced pyridine (flavin) nucleotide.
Pyridine Nucleotide Transhydrogenase.
The transhydrogenation
reaction,
TPNH + DPN+ e DPNH + TPN+, was shown to be catalyzed by an enzyme
obtained from Pseudomonas juorescens (138) and from animal tissues (281).
DifEculties were encountered in demonstrating the reversibility of the reaction with the
bacterial enzyme, until it was found that TPN is a potent inhibitor of the reaction
and that adenosine-2’.phosphate counteracts the inhibition. With the animal enzyme
which is not stimulated by adenosine-2’.phosphate
the equilibrium was found to
be close to unity as expected from the similar values for the redox potential of the
two nucleotides.
SpeciJicity. The bacterial enzyme catalyzes also the hydrogen transfer from
TPNH to desamino TPN and to nicotinamide mononucleotide (NMN).
With
DPNH a transfer reaction to desamino DPN and to NMN was observed. The
animal enzyme catalyzes the transfer from DPNH to desamino DPN, but not from
TPNH to desamino TPN.
An interesting speculation was offered (282) that in animal tissues transhydrogenase, which is usually found associated with the tissue particles, may be concerned
with the transport of hydrogen from bound to free nucleotides. The enzyme may be
of importance for the efficient utilization of free TPNH in coupled phosphorylation
by transferring the hydrogen to bound DPN. It may also function in the opposite
direction, when ‘carrier DPNH’ is needed.
Pyridine-Nucleotide
Flavin-Nucleotide
Transhydrogenase.
These enzymes are
widely distributed in animal tissues, plants and microorganisms. They are usually
quite spectic for a particular pyridine-nucleotide and sometimes specific for a flavinnucleotide. Some of the yellow enzymes are autooxidizable, e.g. the old yellow enzyme (283) ; some require a dye as hydrogen acceptor, e.g. diaphorase (284) ; some
transfer electrons via a metal, e.g. iron in DPNH-cytochrome
c reductase (285).
Little is known in regard to the actual mechanism of electron transfer between the
nucleotides. Though free radicals have been frequently suggested, no direct evidence
for this is available. Since highly purified preparations of both TPNH and DPNH
oxidizing enzymes have been described (286-288) ; a direct study with large amounts
of enzyme should be possible. Changes in the absorption spectrum of DPN-cytochrome reductase in the presence of p-chloromercuribenzoate
have been reported
(285).
Further studies on the chemical oxidation of DPNH by riboflavin compounds
and investigations with deuterium-labeled DPNH may shed further light on these
reactions. An enzyme present in plants (cf. 289, 290) and in yeast (291) was shown
to oxidize DPNH in the presence of ascorbic acid. The yeast enzyme is a flavoprotein which can be resolved and reactivated by either FMN or FAD. A labile
one-electron oxidation product of ascorbic acid, formed by the action of ascorbic
oxidase, was proposed to act as the primary electron acceptor (291).
Votume
E. RACKER
50
35
A detailed discussion of the individual flavoproteins will be omitted since these
enzymes were covered in extensive reviews by Theorell(292) and Singer and Kearney
(119).
CONCLUDING
REMARKS
The impressive number of different DPN-linked enzymes obtainable from various cells emphasizes the general importance of these catalysts. But this is almost
all we dare to say about their physiological function. The demonstration of an enzyme or even a chain of enzymes in a cell free homogenate will not be accepted as
evidence for a physiological pathway by anyone who is aware of the polygamic
nature of nature. Quantitative evaluation of alternate pathways is at its beginning
and is vastly complicated by the uneven distribution of sets of enzymes in the structures of subcellular organization. One of the most important roles of DPN is its
function as hydrogen carrier in biological oxidations. But our knowledge of the intracellular flow of electrons from DPNH is still very incomplete. In liver microsomes a
major hemochromogen with a low redox potential (EJ = -0.12
v. at PH 7.0) has
been found capable of oxidizing DPNH (293). In fact, calculation of the reduction
rate of this new cytochrome (b6) by DPNH casts serious doubts in regard to the
participation of the ‘classical’ flavoproteins in this process (294). Although this
and other iron-porphyrin catalyzed oxidations of DPNH may be of great physiological importance, their discussion must be postponed until they have been further
characterized.
It may be appropriate to close this ‘physiological’ review by quoting from an
address of N. L. Edson (295) :
“In concluding, I ask to be forgiven for relating an unsubstantial tale which
hangs on inadequately tested hypotheses. Since the enzymes are labile and difficult
to purify, some time must pass before the hypotheses meet their fate, and I must
crave a special indulgence of the physiologists because my colleagues and I have been
guilty of destroying enormous numbers of cells in order to trifle with ideas that are
current in biochemistry.”
The author wishes to acknowledge
Srere, H. L. Kornberg
and H. Harbury
the helpful criticism
during the preparation
of Drs. J. S. Fruton,
of the manuscript.
I. Krimsky,
P.
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