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FEMS Yeast Research 2 (2002) 481^494
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MiniReview
The three zinc-containing alcohol dehydrogenases from
baker’s yeast, Saccharomyces cerevisiae
Vladimir Leskovac
a
a;
, Svetlana Trivic¤ b , Draginja Peric›in
a
Faculty of Technology, The University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Yugoslavia
b
Faculty of Science, The University of Novi Sad, 21000 Novi Sad, Yugoslavia
Received 7 February 2002; received in revised form 1 August 2002 ; accepted 2 August 2002
First published online 18 September 2002
Abstract
This review is a summary of our current knowledge of the structure, function and mechanism of action of the three zinc-containing
alcohol dehydrogenases, YADH-1, YADH-2 and YADH-3, in baker’s yeast, Saccharomyces cerevisiae. The opening section deals with the
substrate specificity of the enzymes, covering the steady-state kinetic data for its most known substrates. In the following sections, the
kinetic mechanism for this enzyme is reported, along with the values of all rate constants in the mechanism. The complete primary
structures of the three isoenzymes of YADH are given, and the model of the 3D structure of the active site is presented. All known
artificial mutations in the primary structure of the YADH are covered in full and described in detail. Further, the chemical mechanism of
action for YADH is presented along with the complement of steady-state and ligand-binding data supporting this mechanism. Finally, the
bio-organic chemistry of the hydride-transfer reactions catalyzed by the enzyme is covered: this chemistry explains the narrow substrate
specificity and the enantioselectivity of the yeast enzyme.
5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Keywords : Yeast alcohol dehydrogenase; Alcohol dehydrogenase ; Saccharomyces cerevisiae
1. Introduction
Yeast alcohol dehydrogenase (EC 1.1.1.1) is a member
of a large family of zinc-containing alcohol dehydrogenases. The primary structures of 47 members of this family
have been determined and aligned, and an evolutionary
tree has been constructed, assuming a divergent evolution
from a common ancestral gene [1]. In this way, it was
possible to identify four divergent groups of alcohol dehydrogenases in this family: vertebrates, plants, eukaryotic
microorganisms and prokaryotic bacteria. Baker’s yeast
(Saccharomyces cerevisiae), a member of the third group,
has three isoenzymes of alcohol dehydrogenase : YADH-1,
YADH-2, and YADH-3. YADH-1 is the constitutive form
that is expressed during anaerobic fermentation [2].
YADH-2 is another cytoplasmic form, which is repressed
by glucose [3], and YADH-3 is found in the mitochondria
[4]. YADH-1 accounts for the major part of alcohol dehydrogenase activity in growing baker’s yeast.
The structure, function and mechanism of action of
yeast alcohol dehydrogenase have been reviewed three decades ago [5,6]. The purpose of this article is to update the
subject and to review novel data on the structure, function
and mechanism of action of the isoenzyme YADH-1; this
isoenzyme will be abbreviated as YADH throughout the
text. The steady-state kinetic constants are presented in
the nomenclature of Cleland [7].
2. Isoenzymes of YADH
* Corresponding author. Fax: +381 (21) 350 122.
E-mail address : [email protected] (V. Leskovac).
Abbreviations : YADH, yeast alcohol dehydrogenase, isoenzyme
YADH-1; NDMA, p-nitroso-N,N-dimethylaniline ; AA, acetamide; Az,
sodium azide; DACA, N,N-dimethylamino-trans-cinnamaldehyde
Yeast alcohol dehydrogenase was one of the ¢rst enzymes to be puri¢ed and isolated [8]. If the steady-state
kinetic properties of the ADH isoenzymes are compared, a
large degree of similarity is detected. Table 1 shows the
steady-state kinetic constants for the three isoenzymes of
YADH, isolated from baker’s yeast.
1567-1356 / 02 / $22.00 5 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 7 - 5
FEMSYR 1513 11-11-02
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V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
Table 1
Steady-state kinetic constants of yeast ADH isoenzymes with ethanol
and acetaldehyde as substrates, at pH 7.3, 30‡Ca
Constant
Unit
YADH-1
YADH-2
YADH-3
V1
KA
KB
V1 /KB
V2
KQ
KP
V2 /KP
s31
WM
mM
mM31 s31
s31
WM
mM
mM31 s31
340
170
17
20
1700
110
1.1
1540
130
110
0.81
160
1040
50
0.09
11 550
450
240
12
37.5
2100
70
0.44
4770
a
At neutral pH, the equilibrium is shifted far to the left
(Table 2).
Substrate speci¢city of YADH is restricted to primary
unbranched aliphatic alcohols, and any branching in the
side chain diminishes the activity of the enzyme and lowers
its e⁄ciency. In addition, the enzyme also shows activity
towards secondary alcohols. Table 2 presents the steadystate kinetic constants for various alcoholic substrates and
Table 3 shows the steady-state constants for various carbonyl substrates of the yeast enzyme.
Ethanol is by far the best substrate of the yeast enzyme.
Methanol is a very poor substrate of YADH; the methanol activity of the enzyme at pH 8.8 is only 0.07% of its
ethanol activity under identical conditions. The enzyme is
able to oxidize methanol by NADþ to formaldehyde and
NADH, but the enzymatic reaction is very complex due to
interference of numerous side reactions [18].
Allyl and cinnamyl alcohol are, however, excellent substrates; kinetic constants for the latter alcohol are:
V1 = 133 s31 and V1 /KB = 29 mM31 s31 , at pH 8.2, 25‡C
[19]. (S)-(+)-Butan-2-ol is a much better substrate than
(R)-(3)-butan-2-ol (V1 = 1.0 and 0.05 s31 , and V1 /KB =
18 and 0.8 M31 s31 , respectively, at pH 7.3, 30‡C) [20].
4-Methyl-1-pentanol (V1 = 7 s31 , pH 8.2) is a much better
substrate than 2-methyl-1-propanol (V1 = 0.2 s31 , pH 7.3)
or 3-methyl-1-butanol (V1 = 0.3 s31 , pH 8.2) [19,20].
It was reported that glycerol, glyceraldehyde and acetol
are poor substrates of YADH [21], whereas benzyl alcohol
and benzaldehyde are extremely poor substrates of this
enzyme [20,22]. It has also been reported that p-chlorobenzyl alcohol and p-methoxybenzyl alcohol are slowly oxidized by NADþ in the presence of YADH [23]. 2-Chloroethanol, 2-£uoroethanol, 2,2,2-tri£uoroethanol, propargyl
alcohol, glycidol and polyethylene glycol are no substrates
of the yeast enzyme [24].
Calculated from the data of Ganzhorn et al. [9].
It is evident that YADH-1 and YADH-3 have very
similar kinetic characteristics, while YADH-2 has a
much higher substrate speci¢city for ethanol (V1 /KB ) and
acetaldehyde (V2 /KP ), and much lower Michaelis constants
with ethanol (KB ) and acetaldehyde (KP ). Recently, the
kinetic characterization of YADH-1 and YADH-2 has
been extended by measuring their speci¢city constants
(V1 /KB ) for a number of long-chain alcohols and diols.
It was found that for all alcohols, normalized rates with
YADH-2 were about three-fold faster than with YADH-1
[10].
3. Substrate speci¢city
Yeast alcohol dehydrogenase catalyzes the following reversible redox reaction [5]:
ð1Þ
Table 2
Steady-state kinetic constants for the oxidation of various alcohols at neutral pH
Constant
Unit
Ethanola
Propan1-ola
Butan1-ola
Hexan1-olb
Decan1-olb
Propan2-olc
(S)-(+)-Butan2-olc
Allyl
alcohold
Ethyleneglycold
Trise
V1
KA
KiA
KB
V1 /KA
V1 /KB
0.0001
V1 KiA /KA
Keq f
s31
WM
WM
mM
mM31 s31
mM31 s31
454
109
325
21.7
4165
20.9
67
150
235
29.2
447
22.9
25
250
160
32
100
0.78
15.4
169
152
3.2
91
4.8
14.4
200
190
0.1
72
144
7
597
378
117
11.7
0.06
0.9
376
398
35
2.4
0.026
546
520
730
14.6
1058
37.5
7.0
370
550
444
19.2
0.016
0.5
698
842
6415
0.72
s31
^
1354
0.00019
105
^
16
0.00027
13.8
^
13.7
^
4.4
0.146
0.95
0.40
766
^
10.4
^
0.60
^
a
Calculated from the data of
Calculated from the data of
c
Calculated from the data of
d
Calculated from the data of
e
Calculated from the data of
f
Keq = V1 KiQ KP /(V2 KiA KB ).
b
Dickinson and Monger [11], at pH 7.0, 25‡C.
Scho«pp and Aurich [12], at pH 8.0, 25‡C.
Trivic¤ and Leskovac [13], at pH 7.0, 25‡C.
Trivic¤ and Leskovac [14], at pH 7.0, 25‡C.
Chen and Huang [15], at pH 8.2, 25‡C.
FEMSYR 1513 11-11-02
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
483
Table 3
Steady-state kinetic constants for the reduction of various carbonyl substrates at neutral pH
Constant
Unit
Acetaldehydea
Butyraldehydea
Acetoneb
Butan-2-one-b
Chloroacetaldehydec
NDMAd
DACAe
V2
KQ
KiQ
KP
V2 /KQ
V2 /KP
V2 KiQ /KQ
s31
WM
WM
mM
mM31 s31
mM31 s31
s31
3850
96
12.5
0.9
40100
4280
501
3450
97
7
27.5
35570
125
249
9
43
17.5
477
209
0.019
3.66
0.7
38
15.2
285
18.4
0.0025
0.38
117
270
74
4
431
25.2
31.9
2.1
456
119
1.5
4.5
1.4
0.54
0.176
46
7.6
0.61
3.8
0.29
0.03
a
Calculated
Calculated
c
Calculated
d
Calculated
e
Calculated
b
from
from
from
from
from
the data
the data
the data
the data
the data
of
of
of
of
of
Dickinson and Monger [11], at pH 7.0, 25‡C.
Trivic¤ and Leskovac [13], at pH 7.0, 25‡C.
Leskovac et al. [16], at pH 9.0, 25‡C.
Trivic¤ et al. [17], at pH 8.9, 25‡C.
Leskovac et al. [16], at pH 7.0, 25‡C.
Yeast alcohol dehydrogenase catalyzes three essentially
irreversible chemical reactions:
ClWCH2 WCHO þ NADH þ Hþ ! ClWCH2 WCH2 OH þ NADþ
ð2Þ
ðCH3 Þ2 NWC6 H4 WNO þ 2NADH þ 2Hþ
! ðCH3 Þ2 NWC6 H4 WNH2 þ 2NADþ þ H2 O
ð3Þ
CH3 CHO þ NADþ þ H2 O ! CH3 COOH þ NADH þ Hþ
ð4Þ
Chloroacetaldehyde is an excellent substrate of YADH
(Table 3), while 2-chloroethanol is not oxidized by NADþ ,
which makes the reaction 2 essentially irreversible [16].
p-Nitroso-N,N-dimethylaniline (NDMA) is readily reduced by NADH, in the presence of YADH (reaction
4); the primary product of this reaction, the corresponding
hydroxylamine, is transformed into a quinonediimine
compound by the loss of a molecule of water. The last
compound is reduced non-enzymatically by NADH to
p-amino-N,N-dimethylaniline [17,25]. YADH has a weak
aldehyde dehydrogenase activity; it is able to catalyze
an irreversible oxidation of acetaldehyde to acetic acid
with NADþ , with an apparent kcat = 2.3 s31 and V/K =
34 M31 s31 , at pH 8.8, 22‡C [26].
Free acetaldehyde is a true substrate for alcohol dehydrogenase [27], and gem-diol is probably a true substrate
for aldehyde dehydrogenase activity of YADH [26].
steady-state random mechanism on the alcohol side, and
a steady-state ordered mechanism on the aldehyde side of
the catalytic cycle, with primary aliphatic alcohols and
aldehydes (Scheme 1).
The mechanism in Scheme 1 is restricted to primary
unbranched aliphatic alcohols and aldehydes, if the latter
are present in lower concentrations [33]. The initial rate
equation for this mechanism, in the forward direction and
in the absence of products, is given by [37]:
E0
X
1
1
¼
þ þ
V0
k9 k 5 k7
þ
1 Xk1 k14
k12 þ k13 A þ k11 B
1
W
þ
k1 k9 k13
k12 þ k13 A þ ðk13 =k1 Þk11 B A
1 Xk4
k12 þ k13 A
1
W
þ
þ
k3 k 9
k12 þ k13 A þ ðk13 =k1 Þk11 B B
þ
1 Xk4
k12 þ k13 A þ k11 B
1
W
þ
k3 k 9
k12 þ k13 A þ ðk13 =k1 Þk11 B AB
ð5Þ
where X = 1+k10 /k5 .
The applicability of Eq. 5 to alcohol oxidation is readily
apparent. Eq. 5 predicts that the monomolecular kinetic
constant V1 and the bimolecular speci¢city constants V1 /
KA and V1 /KB are dependent on the nature of substrate B;
inspection of data in Table 2 shows that this is true for all
primary unbranched alcohols. Also, Eq. 5 predicts that the
inhibitory constant KiA is dependent on the nature of substrate B and, therefore, cannot be equal to the dissociation
constant of the EWNADþ -complex ; Table 2 shows that this
is true for all the above alcohols. In addition, a direct
determination of Kþ
E;NAD shows that it is not equal to
4. Steady-state kinetic mechanism
Yeast alcohol dehydrogenase catalyzes the chemical reactions described by Eq. 1. Numerous investigations of the
steady-state kinetic mechanism of the yeast enzyme have
been conducted by several authors [9,11,28^36] ; they have
led to the conclusion that the yeast enzyme follows the
FEMSYR 1513 11-11-02
Scheme 1.
484
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
Table 4
Thermodynamics of the yeast alcohol dehydrogenase reaction, at pH 7.0, 25‡C [45]
k1
k31
k32
k9
k5
k7
k2
k41
k42
k10
k6
k8
E3EA3EA3EAB3EPQ3EQ3E
Rate constant
vG0 (kJ/mol)
Dissociation constant
s
k1
k2
k4 /k3
WM
s31
WM
k9
k10
k5
k6
k7
k8
s31
s31
s31
WM31 s31
s31
WM31 s31
31
31
a
7 X 0.2
2100 X 57
158 000
(11)
(3900)a
(^)a
3980 X 97
35 040 X 870
10 900 X 160
5.0 X 0.04
388 X 5
28.1 X 0.5
(4000)a
(35 000)a
(11 000)a
(4.3)a
(480)a
(44)a
k2 /k1
WM
300
320.08
k41 /k31
k42 /k32
k10 /k9
^
WM
75b
2110c
8.75
10.70
315.26
5.37
k5 /k6
WM
2180
15.18
k7 /k8
WM
13.80
27.73
Total
Keq = 0.000068d
23.64
23.7
a
Data in parentheses are from the steady-state kinetic measurements of Dickinson and Dickenson [31], at pH 7.0, 25‡C.
Taken from Northrop [46].
c
Calculated from the equilibrium constant: k4 /k3 = (k41 /k31 )/(k42 /k32 ).
d
Calculated from the Haldane relationship : Keq = V1 KiQ Kp /(V2 KiA KB ).
b
KiA , in any case (Fig. 4). The initial-rate equation in the
reverse direction, reduction of aldehydes, and in the absence of substrates of reaction, is given by the general
expression for the steady-state ordered mechanism [38] :
E0
Y
1
1
1
Yk5 þ k10 k7 ðYk5 þ k10 Þ
þ
¼
þ þ
þ
þ
V0
k6 k10 P
k2 k4 k10
k8 Q
k6 k8 k10 PQ
ð6Þ
where Y = 1+k9 /k4 .
Eq. 6 satis¢es the results obtained for the reduction of
acetaldehyde and butyraldehyde in predicting a linear reciprocal equation, in which the KiQ , V2 /KQ and V2 KiQ /KQ
constants are independent of the nature of the aldehyde
(Table 3).
The kinetic mechanism in Scheme 1 is compatible with
deuterium isotope e¡ects on maximal rates reported for
ethanol, D V1 = 1.8, D V1 /KA = 1.8, and D V1 /KB = 3.2 [39],
propan-1-ol, D V1 = 3.7 [40], butan-1-ol, D V1 = 3.7 [41],
and propan-2-ol, D V1 = 2.2 around neutrality [13]. With
ethanol, the e¡ect on D V1 /KA was smaller than on D V1 /
KB , suggesting that NADþ binds before ethanol; the still
signi¢cant size of D V1 /KA is probably due to dissociation
of NADþ from the ternary complex [39].
With propan-2-ol and acetone, the kinetic mechanism is
steady-state random in both directions [13]. A similar kinetic mechanism probably holds for most branched and
secondary alcohols [34].
5. Pre-steady-state kinetics
Pre-steady-state kinetic studies provide the numerical
values of the rate constants in the mechanism. The presteady-state kinetics of yeast alcohol dehydrogenase has
been studied with the help of the KINSIM and FITSIM
computer programs of Frieden [42^44].
These computer software packages can simulate the reaction progress curves and calculate the individual rate
constants therefrom (Table 4). The magnitudes of the individual rate constants in Scheme 1 were calculated from
reaction progress curves in both directions, keeping the
concentration of reactants at such a level that dissociation
of NADþ from the central complex was prevented, and
therefore excluding the rate constants k11 ^k14 (Table 4)
[45].
One can see from Table 4 that the magnitudes of rate
constants obtained from the computer simulation of reaction progress curves [45] and from the steady-state kinetics
[31] are very similar, the di¡erences re£ecting only the
di¡erent enzyme preparations.
In the horse liver enzyme, a large conformational
change of the enzyme is triggered when the coenzyme
binds, well documented both in structural terms [47] and
by kinetic methods [48]. Recently, Northrop has reported
that moderate pressure increases the capture of benzyl
alcohol (V1 /KB ) in YADH-catalyzed oxidation of this alcohol with NADþ , by activating the hydride transfer step
[49]. This means that the collision complex for hydride
transfer (*EWNADþ ) has a smaller volume than the free
alcohol plus the capturing form of the enzyme (EWNADþ )
[46]. This was a direct experimental proof for the isomerization step in the yeast enzyme, which enabled the estimation of the equilibrium constant k41 /k31 [75]; using this
value, it was possible to calculate the equilibrium constant
k42 /k32 (Table 4).
Inspection of data in Table 4 clearly shows that, in the
forward direction (oxidation of ethanol at neutral pH), the
rate-limiting step is not the chemical reaction (k9 ), but the
dissociation of NADH from the EQ-complex (k7 ). Like-
FEMSYR 1513 11-11-02
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
wise, NADþ dissociates much faster from the EA-complex
(k2 ) than NADH dissociates from the EQ-complex (k7 ).
6. Primary structure
YADH-1 is a tetramer, composed of four identical subunits ; each subunit consists of a single polypeptide chain
with 347 amino acids, with a molecular mass of 36 kDa
[47]. Each subunit has one coenzyme-binding site and one
¢rmly bound zinc atom, which is essential for catalysis
[50,51] ; the catalytic domain provides the ligands to this
zinc atom: Cys-46, His-67 and Cys-174. The second zinc
atom/subunit is liganded in a tetrahedral arrangement by
four sulfur atoms from the cysteine residues 97, 100, 103
and 111; this zinc atom only has a structural role [52].
Table 5 shows the primary structures of the three isoenzymes of YADH [4,53^55]. The alignment of amino acid
residues for all 47 members of the ADH family was made
progressively rather than pairwise [1].
7. The active site
The amino acid sequences of horse liver alcohol dehy-
Table 5
Primary structure of the three isoenzymes of yeast alcohol dehydrogenase
485
drogenase and YADH-1 are homologous, and the homology amounts to 25% of the amino acid residues [5].
YADH-1 has been crystallized, but only preliminary crystallographic studies have been reported [56]. The threedimensional structure of horse liver alcohol dehydrogenase
in several binary and ternary complexes with coenzymes,
substrates and inhibitors has been solved at high resolution [47]. The tertiary structures of liver and yeast enzyme
are highly similar and able to accommodate extensive sequence changes between the enzymes [57].
Analogous to the liver enzyme, the subunits of the yeast
enzyme are probably divided into two domains : the catalytic domain and the coenzyme-binding domain. The two
domains are unequal in size; the catalytic domain contains
3/5 of all amino acids, whereas the coenzyme-binding domain contains the remaining 2/5 of the amino acids. The
domains are separated by a cleft, containing a deep pocket
which accommodates the substrate and the nicotinamide
moiety of the coenzyme. One domain binds the coenzyme
and the other provides ligands to the catalytic zinc, as well
as to most of the groups that control substrate speci¢city
[47].
Since the liver and yeast enzymes are homologous, molecular modeling of the yeast enzyme can approximate the
structure of one subunit, but not yet the quaternary arrangement [57].
Fig. 1 shows a model of the active site of the yeast
enzyme, drawn schematically after a model obtained in a
molecular graphics display system by Plapp et al. [58]. The
3D-model of the active site of YADH provides an illustration of the main working machinery of the yeast enzyme. In order to perform catalysis, the active site of the
enzyme has to bind a molecule of substrate and a molecule
of coenzyme in a productive mode, and, subsequently catalyze a hydride-transfer reaction between them.
The adenosine-binding site is easily accessible from solution, whereas the nicotinamide-binding site is situated at
the center of the molecule, buried deep inside the protein
[47]. Numerous amino acid residues in the primary structure of the enzyme are involved in substrate and coenzyme
binding and in catalysis (Table 6).
7.1. Substrate-binding pocket
The numbering of amino acids corresponds to horse liver alcohol dehydrogenase; alignment and numbering of amino acid in the yeast isoenzymes according to Sun and Plapp [1].
The inner wall of the pocket is lined with hydrophobic
side chains from the residues Trp-57, Trp-93, Asn-110,
Leu-132, Tyr-140, Thr-141, Met-294, Ala-296, and Ile318, which are from the same subunit as the zinc ligands.
The substrate-binding site near zinc is narrow, because
access is limited by Trp-93 and Thr-48.
The voluminous amino acid side chains of Trp-57, Trp93 and Met-294 make the substrate-binding pocket in the
yeast enzyme much more narrow than the corresponding
pocket in the horse liver enzyme.
FEMSYR 1513 11-11-02
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V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
7.2. Ligands to the active-site zinc
At the bottom of the substrate-binding pocket, a zinc
atom is coordinated to three protein ligands: two thiolates
from Cys-46 and Cys-174, and one imidazole nitrogen of
His-67. The other imidazole nitrogen of His-67 is hydrogen-bonded to a carboxylic group of Asp-49. A carboxylic
group of Glu-68 is also in close proximity to the active-site
zinc atom. Asp-49 and Glu-68 are the residues conserved
in all known zinc-dependent alcohol dehydrogenases; instead of being inner-shpere ligands to the zinc, both amino
acids are situated in the second sphere. The only polar
groups in the pocket close to zinc are the zinc ligands,
the nicotinamide moiety of the coenzyme, and the side
chain of Thr-48.
7.3. Nicotinamide ring
The nicotinamide ring binds in a cleft in the interior of
the protein, close to the center of the molecule. On one
side the ring interacts with Thr-178, Leu-203 and Met-294.
The other side faces the active site, and is close to the
catalytic zinc atom and the sulfur ligands of Cys-46 and
Cys-174.
The oxygen atom of the carboxamide group is hydrogen-bonded to the main-chain nitrogen atom of Val-319.
The nitrogen atom of the carboxamide group is hydrogenbonded to the carboxyl oxygens of Val-292 and Ser-317.
The side chain of Thr-178 helps to keep the nicotinamide
ring of the nucleotide in the correct stereochemical position for hydride transfer (Fig. 5); Thr-178 is conserved in
all known homologous alcohol dehydrogenases.
Fig. 1. Model of the active site of yeast alcohol dehydrogenase, drawn
schematically after Plapp et al. [58].
7.4. The proton-relay system
It was proposed by Eklund et al. [59] that the hydrogenbonded relay system in the liver enzyme (Fig. 3),
Table 6
Positions of residues that participate in enzymatic functions of the yeast
enzyme (adopted from Jo«rnval et al. [57])
Adenine binding pocket
Interior
Ser-198
Ile-250
Ile-222
Ser-269
Gly-224
Ala-274
Phe-243
Ala-277
Surface
Ser-271
Ala-273
Adenosine^ribose binding
Gly-199
Lys-228
Asp-223
Ser-269
Gly-225
Pyrophosphate binding
His-47
Leu-203
Gly-202
Nicotinamide ribose
Gly-293
Ser-269
Nicotinamide
Thr-178
Substrate-binding pocket
Trp-57
Thr-141
Trp-93
Met-294
Asn-110
Ala-296
Leu-132
Ile-318
Tyr-140
Proton relay system
Thr-48
His-51
His 51::::NADþ ::::Ser 48::::CH 2 OHðH 2 OÞ::::Zn2þ ;
stretching from His-51 on the surface of the enzyme to
the active-site zinc atom in the interior of the enzyme,
serves as a proton conductor which helps the dissociation of alcohol to alcoholate in the productive ternary
enzymeWNADþ Walcohol-complex. The yeast enzyme has
the same proton relay system with, however, Ser substituted with Thr (Fig. 1) [58]. Therefore, since the
enzymeWNADþ Walcoholate-complex is considered a true
transition state in the yeast enzyme catalysis [60], the
proton relay system must greatly accelerate the same.
7.5. Binding of the coenzyme
Ligands to active-site zinc atom
Inner sphere
Cys-46
Cys-174
His-67
Second sphere
Asp-49
Glu-68
The coenzyme is bound to the apoenzyme by numerous
secondary valence forces. Important amino acid residues
are: Asp-223, which is hydrogen-bounded to AMP-ribose,
His-47, forming a salt bridge with AMP-orthophosphate,
and Leu-203, forming a hydrogen bond to NMN-orthophosphate.
FEMSYR 1513 11-11-02
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487
8. Mutations in the yeast enzyme
8.2. Ligands to the active-site zinc (Asp-49, Glu-68)
The three yeast ADH genes have been cloned and described [4,54,55]. Therefore, it was possible to change individual amino acids in the primary structure of YADH-1
via site-directed mutagenesis and isolate a quantity of mutated enzymes (Table 7).
In recent years, a number of genetically engineered mutants of YADH-1 were isolated and kinetically characterized, principally by Plapp and his co-workers. Most of
these mutations involve amino acids which are intimately
involved in the binding of substrates and in catalysis, and
provide information about the general principles concerning the function of the catalytic residues. Table 7 shows
the steady-state kinetic properties of all YADH mutants
described so far participating in substrate binding and in
catalysis.
The carboxylate group of Asp-49 is hydrogen-bonded to
His-67, which in turn coordinates the active-site zinc ; in
addition, the carboxylate group of Glu-68 is in the vicinity
of the active-site zinc. If Asn is substituted for Asp-49 or
Gln for Glu-68, a negative charge is removed from the
vicinity of the active-site zinc; these substitutions reduce
the catalytic e⁄ciency with ethanol (V1 /KB ) 1000 times
and 100 times, respectively, and the catalytic constant
(V1 ) 40 times. These reductions in activity were interpreted
by an increased electrostatic potential near the active-site
zinc, due to removal of negative charges; as a consequence
the activity is decreased by hindering isomerizations of
enzyme^substrate complexes [39].
8.1. Substrate-binding pocket (Met-294, Trp-57, Trp-93)
An exchange of Ser for Thr-48 does not interrupt the
hydrogen bonding in the proton relay system and, as expected, the activity of the Thr48Ser mutant is very similar
to that of the wild-type. The double mutant Thr48Ser:
Trp93Ala and the triple mutant Thr48Ser:Trp57Met :
Trp93Ala show decreased activities that are obviously
due to removal of bulky tryptophan residues from the
substrate-binding pocket [20]. An exchange of Cys or
Ala for Thr-48 disrupts the hydrogen bonding in the relay
system and, as expected, renders the enzyme inactive [58].
The role of His-51 in catalysis has been tested by replacing it with glutamine or glutamic acid [58,61]. These residues have an appropriate size to form the hydrogen bond
with the 2P-hydroxyl group of the nicotinamide ribose;
thus, binding of the coenzyme in the mutant enzymes
could resemble binding in the wild-type enzyme. On the
other hand, a glutamine residue would not be able to
An exchange of Leu for Met-294, on the edge of the
substrate-binding pocket, has very little in£uence on the
steady-state kinetic properties of the enzyme with ethanol
or acetaldehyde. On the other hand, the Met294Leu mutant has a 10-fold lower catalytic activity (V1 ) with butan1-ol, indicating that the C4-atom of butan-1-ol is in a close
contact with Met-294, whereas the shorter ethanol is not
[9]. An exchange of Met or Leu for Trp-57 decreases the
catalytic e⁄ciency (V1 /KB ) with ethanol only three- to
four-fold, whereas an exchange of Ala for Trp-93 decreases the catalytic e⁄ciency 300-fold; with an enlargement of the substrate-binding pocket in the latter case, the
enzyme acquires weak activity with branched-chain alcohols (2-methyl-1-butanol, 3-methyl-1-butanol) and benzyl
alcohol [19,20].
8.3. The proton-relay system (Thr-48, His-51)
Table 7
Steady-state kinetic constants for YADH mutants, with ethanol and acetaldehyde as substrates, determined at pH 7.3, 30‡C
Mutant
V1 (s31 ) V1 /KA (mM31 s31 )
V1 /KB (mM31 s31 )
V2 (s31 ) V2 /KQ (mM31 s31 )
V2 /KP (mM31 s31 )
Source
YADH-1
Substrate-binding pocket
Met294Leu
Trp57Met
Trp57Leua
Trp93Ala
Ligands to the active-site zinc
Asp49Asn
Glu68Gln
The proton relay system
Thr48Ser
Thr48Ser :Trp93Ala
Thr48Ser :Trp57Met:Trp93Ala
Thr48Cys
Thr48Ala
His51Gln
His51Glu
340
2000
20
1700
15 500
1545
[9]
500
220
99
110
794
265
91
48
26.3
4.9
7.4
0.07
2100
1900
211
ND
26 250
6 790
2 245
ND
2100
513
ND
ND
[9]
[20]
[19]
[20]
7.5
9.9
0.83
24
0.02
0.24
113
730
125
4 560
2.3
13
[39]
[39]
200
140
120
61
61
27
2
2200
11.8
152
0.033
22.2
0.75
No detectable activity
No detectable activity
245
1.4
26
0.26
1500
530
ND
13 640
4 077
ND
2027
5.7
ND
2800
ND
25 450
ND
215
ND
[20]
[20]
[20]
[58]
[58]
[61]
[58]
ND = not determined.
a
Determined at pH 8.2, 25‡C.
FEMSYR 1513 11-11-02
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V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
participate in base catalysis, whereas a glutamate residue
could accept a proton. Plapp et al. [58] have found that a
wild-type enzyme has a distinct pKa value of 7.7 in the
pH-pro¢le for the V1 /KB function. Replacement of His-51
with Gln or Glu reduces the value of V1 /KB 13-fold and
60-fold at pH 7.3, respectively; in addition, the pKa value
of 7.7 in the pH pro¢le of the V1 /KB function is abolished
in both cases. These results were interpreted by a mechanism in which the amino acid residue in the mutant enzyme hinders the deprotonation of alcohol through the
proton relay system [58]. On that interpretation, these results are consistent with the role of His-51 in the proton
relay system, where it participates as a base.
to the free enzyme (k8 ) decreases in alkaline over a single
pKa value 7.8, while the dissociation rate constant for the
EWNADH-complex (k7 ) is almost pH-independent, from
pH 6.5 to 9.0.
In recent years, a number of genetically engineered mutants of YADH-I, with mutations in the adenylate-binding
pocket, have been isolated and kinetically characterized,
principally by Plapp and his co-workers (Table 8). The
following general conclusions may be drawn from the kinetic data shown in Table 8.
9.1. Adenine site substitutions (Ser-198, Gly-224, Gly-225)
Gly224Ile and Gly225Arg mutants have only modest
e¡ects on coenzyme binding and other kinetic constants,
but the Ser198Phe mutant signi¢cantly decreases its a⁄nity for coenzymes and turnover numbers.
9. Binding of coenzymes
Fig. 2 summarizes the steady-state and ligand-binding
data relevant for the binding of coenzymes to the free
enzyme.
The dissociation constant of the EWNADþ complex for
the yeast enzyme, Kþ
E;NAD , is practically pH-independent;
on the other hand, the dissociation constant of the
EWNADH complex, KE;NADH , decreases with lower pHvalue over three apparent pKa values (6.6, 8.0 and 9.0).
The association rate constant for the binding of NADH
9.2. Adenosine^ribose binding (Asp-223)
The Asp223Gly:Gly225Arg double mutant shows a decrease in all kinetic parameters, but uses NAD(H) and
NADP(H) with about the same e⁄ciency.
9.3. Pyrophosphate binding (His-47, Ala-200, Leu-203,
Gly-204)
Mutation of the residues Ala-200, Leu-203 or Gly-204
decreases all kinetic parameters signi¢cantly, suggesting
that these amino acids are essential for the binding of
the pyrophosphate moiety of the coenzyme. On the other
hand, substitution of His-47 by the basic amino acid Arg
decreases the catalytic activity with NAD(H) only modestly.
9.4. Nicotinamide^ribose binding (Ser-269)
The Ser269Ile mutant decreases its turnover numbers by
350-fold.
Studies of the mutants in the adenylate-binding site of
the enzyme show that several amino acid residues at the
proposed adenylate-binding site of the enzyme are important for coenzyme binding and formation of productive
ternary complexes. The Asp223Gly:Gly225Arg double
mutant was the only mutant that uses NAD(H) and
NADP(H) with about the same e⁄ciency ; this result suggests that conversion of the coenzyme speci¢cally requires
multiple substitutions [62]. Mutations of amino acids Leu203 and Thr-178 have been performed in order to locate
the structural determinants of the high stereospeci¢city of
the enzyme for the coenzyme NAD(H) [64].
Fig. 2. pH pro¢les for the binding parameters of coenzymes to the free
enzyme; rate constants k7 and k8 , as in Scheme 1 (adopted from Leskovac et al. [34]).
10. Chemical mechanism
Primary structure, tertiary structure and point muta-
FEMSYR 1513 11-11-02
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489
Table 8
Steady-state kinetic constants for YADH mutants in the adenylate binding pocket, with ethanol and acetaldehyde as substrates, determined at pH 7.3,
30‡C
Mutant
V1 (s31 )
V1 /KA (mM31 s31 )
V1 /KB (mM31 s31
V2 (s31 )
V2 /KQ (mM31 s31 )
V2 /KP (mM31 s31 )
Source
YADH-1
Adenine site substitutions
Ser198Phe
Gly224Ile
Gly225Arg
Adenosine^ribose binding
Asp223Gly
Asp223Gly:Gly225Arg
Pyrophosphate binding
Leu203Alaa
Leu203Ala :Thr178Sera
His47Arg
Gly204Ala
Ala200v:Ala201Leub
NMN^ribose binding
Ser269Ile
340
2000
20
1700
15 500
1 545
[9]
40
360
550
1.25
414
1222
0.14
16
21
150
4000
2400
25
6 060
12 000
71
20 000
18 000
[62]
[62]
[62]
38
17
2.1
0.94
0.2
0.13
300
110
60
18.33
75
20
[63]
[63]
106
31.9
60
8
67
56.4
61.3
400
0.26
13.13
ND
ND
0.9
0.02
1.4
ND
ND
460
200
ND
ND
ND
46 000
25
ND
ND
ND
98
11
ND
[64]
[64]
[32]
[62]
[62]
1.0
0.36
0.003
5.4
13.85
0.31
[62]
ND = not determined.
a
Determined at pH 8.2, 25‡C.
b
Alignment of amino acids according to Table 5. Ala200 is an insertion in the yeast enzyme with respect to other members of the alcohol dehydrogenase family of enzymes; therefore, this residue is not counted in the primary structure that follows after this residue [1].
tions in the yeast enzyme, outlined in the preceding sections, strongly suggest that the integrity of the proton
relay system is indispensable for the activity of the enzyme.
Based on this integrity of the relay system, which is
maintained throughout the catalytic cycle, Cook and Cleland [60] have proposed the chemical mechanism of action
for the yeast enzyme as shown in Scheme 2.
In this mechanism, B and P represent alcohol and ketone, and k3 , k4 , k5 and k6 represent hydride-transfer
steps ; X is an intermediate with the stoichiometry of an
alkoxide, and k1 and k2 are the steps in which a proton is
transferred from B to a group on the enzyme to give X,
and similarly for the reverse process.
An assignment of appropriate pKa values to all dissociation forms of the enzyme in Scheme 2 was founded on
studies of the pH dependence of the steady-state kinetics
and ligand-binding parameters [14,16,26,33^36,65], as outlined below.
Table 9 shows the macroscopic pKa values calculated
from the pH pro¢les of the maximal rates (V1 ) and the
speci¢city constants (V/K) with various substrates.
Table 10 presents the pKa values calculated from the pH
pro¢les of binding constants (Ki ) for competitive dead-end
inhibitors.
Scheme 2.
The speci¢city constants V/K with ‘nonsticky’ substrates, such as propan-1-ol, propan-2-ol, NDMA,
DACA and acetone, provide information on catalytically
active groups in enzyme^coenzyme complexes [66], if the
pH pro¢les of V/K are ¢tted to initial-rate equations appropriate to the mechanism in Scheme 2 [36]. In this way,
the pK1 (8.0) and pK5 (7.9^8.0) values in Scheme 2 were
estimated. From the binding of azide, a dead-end inhibitor
competitive with alcohols, the value for pK1 (7.9) was
con¢rmed; from the binding of acetamide, a dead-end
inhibitor competitive with aldehydes, the values for pK4
(8.3) and pK5 (7.9) were estimated.
pH pro¢les for the V1 function provide information on
catalytically active groups in the productive ternary enzymeWNADþ Walcohol-complex [66]. In this way the pK2
value was estimated (8.3), from the pH pro¢les of V1
with butan-1-ol and propan-2-ol.
An indirect estimation provided the value of pK3 (8.3)
[36].
The chemical mechanism of action, presented in Scheme
1, can be drawn entirely in terms of the proton relay
system, as is shown in Fig. 3; in Fig. 3, however, the
Thr-48 residue was omitted from the relay for the sake
of simplicity. The key feature of Fig. 3 is that His-51 lies
at the surface of the protein and thus can be deprotonated
as in the conversion of HEAX to EAX or HEQP to EPQ,
while reactants are bound and the state of protonation of
molecules in the substrate-binding site is locked. Thus,
HEAX can be deprotonated to EAX without preventing
subsequent hydride transfer.
A di¡erent view on the chemical mechanism of action of
yeast alcohol dehydrogenase has been presented by Bra«nde¤n et al. [5]. These authors proposed that the Zn2þ -bound
FEMSYR 1513 11-11-02
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V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
Table 9
Macroscopic pKa values and pH-independent limiting constants in various YADH-catalyzed reactions (adopted from Leskovac et al. [36])
Substrate
pKa
Butan-1-ol
V1 (s31 )
6.1
7.3
8.3
Propan-2-ol
6.2
V1 /KB (s31 )
7.4
8.3
Propan-1-ol
6.7
V1 /KB (mM31 s31 ) 7.4
8.2
Propan-2-ol
6.5
V1 /KB (M31 s31 )
7.1
7.8
Acetone
7.9
V1 /KB (M31 s31 )
8.2
9.0
DACA
8.0
V2 /KP (mM31 s31 )
8.0
NDMAa
V2 /KP (mM31 s31 )
Limiting constant
Dixon^Webb plot
191
increases with pH
81
increases with pH
9.0
increases with pH
155
increases with pH
6.9
decreases with pH
0.25
decreases with pH
2.2
0.9
plateau at low pH
plateau at high pH
water dissociates when the coenzyme NADþ is added; the
remaining (OH)3 deprotonates the alcohol, which is then
bound to the Zn2þ ion as the fourth ligand. Fig. 4 shows
this dissociation in the proton relay system.
Recently, Nadolny and Zundel [67] have claimed experimental evidence supporting the above mechanism. These
authors obtained Fourier-transform infrared (FTIR) spectra of various complexes of yeast alcohol dehydrogenase
with NADþ and coenzyme analogs ; from the FTIR spectra they concluded that, upon binding of NADþ to the
enzyme, N1 of the coenzyme adenosine becomes protonated and the molecule of water in the active site dissociates
to a hydroxyl anion. It was postulated that the positive
charge is conducted from the zinc-bound water to histidine-51 and then further to the N1-atom of the adenine
rest via the proton relay system through the protein. Thus
the binding of NADþ to the enzyme shifts the equilibrium
1C2 in Fig. 4 to the right. The substrate, alcohol, is then
deprotonated by the (OH)3 bound to the Zn2þ ion and
forms the structure 3.
The experiments of Nadolny and Zundel [67] with the
yeast enzyme were conducted at pH 7.5 and they do not
explain the pH dependence of the enzyme activity. Further, the proposed mechanism lacks the explanation for
the conductance of the positive charge from His-51 to
\.
adenine across a distance of approximately 7 A
hydrogen (pro-R or A-type) at the 4-position of NADH to
the carbonyl carbon of the substrate (Fig. 5).
The stereochemical ¢delity of the hydride transfer reaction is very high, and YADH makes but one stereochemical ‘mistake’ every 7 000 000 turnovers. If the bulky side
chain of Leu-203 is exchanged with Ala, the Leu203Ala
mutant (Table 7) makes one stereochemical ‘mistake’
every 850 000 turnovers with NADH, and every 450 turnovers with thio-NADH, which has a weaker hydrogenbonding capacity. From this, it was concluded that the
decrease in stereochemical ¢delity comes from an increase
in the transfer rate of the 4-Si-hydrogen of NADH. The
nicotinamide ring of the coenzyme is kept in a correct
position for hydride transfer mainly by hydrogen bonds
between its amide group and Val-292 and Val-319, and the
rotation of 180‡ around the glycosidic bond is obstructed
mainly by the side chain of Leu-203 [64].
The main reaction catalyzed by alcohol dehydrogenase
is, in principle, a very simple reaction. An alcohol group is
oxidized by the removal of a proton from the hydroxyl
group and by the transfer of a hydride ion from the adjacent carbon atom to NADþ . By analogy with the horse
liver enzyme [47], we may assume that hydride transfer in
the yeast enzyme occurs in a completely water-free environment. Direct transfer of a hydride ion is facilitated in a
hydrophobic environment, where water is excluded. The
positive charge on the nicotinamide ring is crucial for the
enhanced binding of alcohol to the enzyme ; insertion of
the positive charge in this hydrophobic environment facilitates formation of the negatively charged alcoholate ion.
The creation of an alcoholate ion greatly facilitates hydride transfer. The important role of the zinc atom in
alcohol oxidation is to stabilize the alcoholate ion for
the hydride-transfer step. In the reverse direction, zinc
functions as an electron attractor, which gives rise to an
increased electrophilic character of the aldehyde, consequently facilitating the transfer of a hydride ion to the
aldehyde. Thus, the proposed mechanism is essentially
electrophilic catalysis mediated by the active-site zinc
atom.
The overall oxidation of alcohol to aldehyde involves
the net release of one proton (Eq. 1); the ultimate source
of this proton is alcohol. The release of a proton from the
bound alcohol occurs in the center of the enzyme molecule
in a region that is inaccessible to solution ; the proton is
transferred by certain groups on the enzyme to the surrounding solution (Fig. 1). Because water is not directly
Table 10
Macroscopic pKa values and pH-independent constants for ternary complexes of YADH with competitive dead-end inhibitorsa
11. Hydride transfer
Complex
One of the classical aspects of coenzyme binding to
yeast alcohol dehydrogenase is the A-stereospeci¢city of
the coenzyme [68]. YADH-catalyzed reactions are highly
stereospeci¢c ; the enzyme catalyzes the transfer of the Re-
þ
þ
EWNAD +Az1EWNAD WAz
EWNADH+AA1EWNADHWAA
a
pKa
Limiting constant
7.9
8.3
0.95 mM (at low pH)
45.8 mM (low pH)
118 mM (high pH)
Calculated from the data of Leskovac et al. [35].
FEMSYR 1513 11-11-02
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
491
Fig. 3. The chemical mechanism of action of alcohol dehydrogenase [36].
involved in the catalytic reaction, that is, no hydrolysis or
hydration, there is no reason to suggest a role for a water
molecule at the active site of YADH [36].
In catalysis, the molecules of the substrate and the nicotinamide ring of the coenzyme probably do not have
¢xed positions. The rearrangement of electron con¢guration on the carbon atom from the sp2 hybridization in
aldehyde to the sp3 in alcohol, requires di¡erent pathways
for hydride transfer and, consequently, di¡erent relative
orientations [69].
Primary and secondary kinetic isotope e¡ects (kH /kD ,
kH /kT and kD /kT ) in YADH-catalyzed reactions have
been studied as a probe of quantum-mechanical hydrogen
tunneling. Hydrogen tunneling was ¢rst suggested in
YADH-catalyzed reactions following the measurement of
anomalously large secondary kinetic isotope e¡ects [70]. In
the absence of tunneling and coupled motion, a secondary
kinetic isotope e¡ect is expected to be intermediate between unity and the value of the equilibrium isotope e¡ect
[71]. Thus, Cook et al. [70] studied the oxidation of 2propanol with [4-2 H]NADþ ,
½4 2 HNADþ þ CH3 CHðOHÞCH3
! ½4S 2 HNADH þ CH3 COCH3 þ Hþ
ð7Þ
and found a secondary kinetic isotope e¡ect of 1.22 and
an equilibrium isotope e¡ect of 0.89, which is indicative of
hydrogen tunneling.
In hydride-transfer reactions catalyzed by YADH, one
can measure the kH /kD , kH /kT , and kD /kT primary, and,
similarly, the secondary isotope e¡ects. The Swain^Schaad
relationship states that, in the absence of tunneling and
coupled motion, and without kinetic complexities,
ðkD =kT Þ3:30 ¼ kH =kT
ð8Þ
Klinman [72] has argued that, out of various thermody-
namic and kinetic criteria, the most sensitive one for detection of tunneling is the breakdown of the above relationship, particularly for the secondary kinetic isotope
e¡ect [73]. In line with this, Cha et al. [74] have studied
the oxidation of benzyl alcohol by NADþ , using six di¡erently labeled alcohols as substrates :
½14 CC 6 H 5 CH 2 OH; ½14 CC 6 H 5 CD2 OH;
C 6 H 5 CðH; TÞOH; C 6 H 5 CðT; HÞOH;
C 6 H 5 CðD; TÞOH; C 6 H 5 CðT; DÞOH
in order to obtain all combinations of isotope e¡ects. This
reaction is suitable for exploring hydrogen tunneling because of the lack of any kinetic complexity, as it has a
rate-limiting H-transfer step [75].
Cha et al. [74] have demonstrated that, in this reaction,
the exponents in Eq. 8 are 3.58 and 10.2 for the primary
and secondary kinetic isotope e¡ect, respectively, indicating signi¢cant breakdown of the semi-classical upper limit.
For hydrogen tunneling to occur, the reactive carbon
atoms have been brought close together so that the classical energy barrier is penetrated. Thus, it appears that
hydrogen tunneling is an additional general phenomenon
which facilitates the YADH catalysis [23,76^78].
Leskovac et al. [79] have studied the primary kinetic
isotope e¡ects and the internal thermodynamics of the
YADH-catalyzed oxidation of 2-propanol-h8 and 2-propanol-d8 with NADþ ; the properties of this reaction were
compared with non-enzymatic model redox reactions of
N1 -substituted-1,4(1 H2 )dihydronicotinamides and N1 -substituted-1,4(1 H2 H)dihydronicotinamides with a number of
various oxidizing agents. The kinetic and thermodynamic
properties of the enzymatic reaction closely resemble the
model hydride-transfer reactions which probably proceed
via a linear transition state, and are very dissimilar from
Fig. 4. The Bra«nde¤n mechanism.
FEMSYR 1513 11-11-02
492
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
[8]
[9]
[10]
[11]
[12]
[13]
Fig. 5. Stereospeci¢city of YADH catalysis. NADH binds anti, presenting Re-hydrogen (HRe ) to acetaldehyde lying above the coenzyme in this
diagram. For clarity, Thr-178 is not shown; the methyl group of this
side chain lies below and to the left of the nicotinamide behind Leu-203
(reproduced from Weinhold et al. [64], with permission of the corresponding author).
[14]
[15]
[16]
reactions which proceed via a bent transition state, suggesting that this particular enzymatic reaction has a linear
transition state.
[17]
[18]
Acknowledgements
[19]
This work was ¢nancially supported by the Ministry of
Science and Technology of the Republic of Serbia.
[20]
References
[21]
[1] Sun, H.-W. and Plapp, B.V. (1992) Progressive sequence alignment
and molecular evolution of the Zn-containing alcohol dehydrogenase
family. J. Mol. Evol. 34, 522^535.
[2] Young, E.T., Williamson, V., Taguchi, A., Smith, M., Sledziewski,
A., Russel, D., Ostermann, J., Denis, C., Cox, D. and Beier, D.
(1982) The alcohol dehydrogenase genes from the yeast, Saccharomyces cerevisiae : isolation, structure and regulation. In: Genetic Engineering of Microorganisms for Chemicals (Hollaender, A., DeMoss,
R.D., Kaplan, S., Konisky, J., Savage, D. and Wolfe, R.S., Eds.),
pp. 335^361. Plenum, New York.
[3] Wills, C. (1979) Amino acid substitutions in two functional mutants
of yeast alcohol dehydrogenase. Nature 279, 734^736.
[4] Young, E.T. and Pillgrim, D. (1985) Isolation and DNA sequence of
ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol
dehydrogenase in Saccharomyces cerevisiae. Mol. Cell Biol. 5, 3024^
3034.
[5] Bra«nde¤n, C.-I., Jo«rnvall, H., Eklund, H. and Furugren, B. (1975)
Alcohol dehydrogenases. In: The Enzymes, Vol. 11, 3rd edn. (Boyer,
P.D., Ed.), pp. 104^190. Academic Press, New York.
[6] Klinman, J.P. (1981) Probes of mechanism and transition-state structure in the alcohol dehydrogenase reaction. CRC Crit. Rev. Biochem.
10, 39^78.
[7] Cleland, W.W. (1963) The kinetics of enzyme-catalyzed reactions
[22]
[23]
[24]
[25]
[26]
[27]
with two or more substrates or products. Biochim. Biophys. Acta
67, 103^137.
Negelein, E. and Wul¡, H.-J. (1937) Diphosphopyridinproteid Alcohol, Acetaldehyd. Biochem. Z. 293, 351^389.
Ganzhorn, A.J., Green, D.W., Hershey, A.D., Gould, R.M. and
Plapp, B.V. (1987) Kinetic characterization of yeast alcohol dehydrogenase. Amino acid residue 294 and substrate speci¢city. J. Biol.
Chem. 262, 3754^3761.
Dickinson, F.M. and Dack, S. (2001) The activity of yeast ADH I
and ADH II with long chain alcohols and diols. Chem. Biol. Interact.
130^132, 417^423.
Dickinson, F.M. and Monger, G.P. (1973) A study of the kinetics
and mechanism of yeast alcohol dehydrogenase with a variety of
substrates. Biochem. J. 131, 261^270.
Scho«pp, W. and Aurich, H. (1976) Kinetics and reaction mechanism
of yeast alcohol dehydrogenase with long chain primary alcohols.
Biochem. J. 157, 15^22.
Trivic¤, S. and Leskovac, V. (1994) Kinetic mechanism of yeast alcohol dehydrogenase activity with secondary alcohols and ketones. Indian J. Biochem. Biophys. 31, 387^391.
Trivic¤, S. and Leskovac, V. (1999) Novel substrate of yeast alcohol
dehydrogenase - 4. Allyl alcohol and ethylene glycol. Biochem. Mol.
Biol. Int. 47, 1^8.
Chen, D.-H. and Huang, T.-C. (1994) Oxidation of Tris (hydroxymethyl) aminomethane by yeast alcohol dehydrogenase. J. Chem.
Eng. Jpn. 27, 547^550.
Leskovac, V., Trivic¤, S., Zeremski, J., Stanc›ic¤, B. and Anderson,
B.M. (1997) Novel substrates of yeast alcohol dehydrogenase - 3.
4-Dimethylamino-cinnamaldehyde and chloroacetaldehyde. Biochem.
Mol. Biol. Int. 43 (007), 365^373.
Trivic¤, S., Leskovac, V., Peric›in, D. and Winston, G.W. (2002) A
novel substrate for yeast alcohol dehydrogenase - p-nitroso-N,N-dimethylaniline. Biotechnol. Lett. 24, 807^812.
Mani, J.-C., Pietruszko, R. and Theorell, H. (1970) Methanol activity
of alcohol dehydrogenase from human liver, horse liver, and yeast.
Arch. Biochem. Biophys. 140, 52^59.
Weinhold, E.G. and Benner, S.A. (1995) Engineering yeast alcohol
dehydrogenase. Replacing Trp 54 by Leu broadens substrate speci¢city. Protein Eng. 8, 457^461.
Green, D.W., Sun, H.-W. and Plapp, B.V. (1993) Inversion of the
substrate speci¢city of yeast alcohol dehydrogenase. J. Biol. Chem.
268, 7792^7798.
Bergmeyer, H.U. (1970) Alkohol dehydrogenase (aus Hefe). In:
Methoden der enzymatischen Analyse, Vol.1, 2nd edn., pp. 392^
393, 1457^1474. Verlag-Chemie, Weinheim.
Scharschmidt, M., Fisher, M.A. and Cleland, W.W. (1984) Variation
of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase
with benzyl alcohol and yeast alcohol dehydrogenase with benzaldehyde. Biochemistry 23, 5471^5478.
Rucker, J., Cha, Y., Jonsson, T., Grant, K.L. and Klinman, J.P.
(1992) Role of internal thermodynamics in determining hydrogen
tunneling in enzyme-catalyzed hydrogen transfer reactions. Biochemistry 31, 11489^11499.
Trivic¤, S., Stanc›ic¤, B. and Leskovac, V. (1997) Oxidation of alcohols
by yeast alcohol dehydrogenase (review). Chem. Ind. (Beograd) 51,
113^118.
Leskovac, V., Trivic¤, S. and Anderson, B.M. (1996) Yeast alcohol
dehydrogenase catalyzed reduction of p-nitroso-N,N-dimethylaniline
by NADH. Ital. J. Biochem. 45, 9^18.
Trivic¤, S., Leskovac, V. and Winston, G.W. (1999) Aldehyde dismutase activity of yeast alcohol dehydrogenase. Biotechnol. Lett. 21,
231^234.
Mu«ller-Hill, B. and Wallenfels, K. (1964) Mechanismus der Wassersto¡-u«bertragung mit Pyridinnucleotiden. XXV. Ist Acetaldehyd oder
sein Hydrat Substrat der Alkoholdehydrogenase ? Biochem. Z. 339,
349^351.
FEMSYR 1513 11-11-02
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
[28] Wratten, C.C. and Cleland, W.W. (1963) Product inhibition on yeast
and liver alcohol dehydrogenase. Biochemistry 2, 935^940.
[29] Silverstein, E. and Boyer, P.D. (1964) Equilibrium reaction rates and
the mechanisms of liver and yeast alcohol dehydrogenase. J. Biol.
Chem. 239, 3908^3914.
[30] Dickenson, C.J. and Dickinson, F.M. (1978) Inhibition by ethanol,
acetaldehyde and tri£uoroethanol of reactions catalysed by yeast and
horse liver alcohol dehydrogenases. Biochem. J. 171, 613^627.
[31] Dickinson, F.M. and Dickenson, C.J. (1978) Estimation of rate and
dissociation constants involving ternary complexes in reactions catalyzed by yeast alcohol dehydrogenase. Biochem. J. 171, 629^637.
[32] Gould, R.M. and Plapp, B.V. (1990) Substitution of arginine for
histidine-47 in the coenzyme binding site of yeast alcohol dehydrogenase. Biochemistry 29, 5463^5468.
[33] Trivic¤, S. and Leskovac, V. (1994) Kinetic mechanism of yeast alcohol dehydrogenase with primary aliphatic alcohols and aldehydes.
Biochem. Mol. Biol. Int. 32, 399^407.
[34] Leskovac, V., Trivic¤, S. and Anderson, B.M. (1996) Use of pH studies to determine the kinetic and chemical mechanism of yeast alcohol
dehydrogenase with primary aliphatic alcohols and aldehydes. Indian
J. Biochem. Biophys. 33, 177^183.
[35] Leskovac, V., Trivic¤, S. and Anderson, B.M. (1998) Use of competitive dead-end inhibitors to determine the chemical mechanism of
action of yeast alcohol dehydrogenase. Mol. Cell. Biochem. 178,
219^227.
[36] Leskovac, V., Trivic¤, S. and Anderson, B.M. (1999) Comparison of
the chemical mechanisms of action of yeast and equine liver alcohol
dehydrogenase. Eur. J.Biochem. 264, 840^847.
[37] Dalziel, K. (1958) Puri¢cation of yeast alcohol dehydrogenase. Trans.
Faraday Soc. 54, 1247^1253.
[38] Segel, I.H. (1975) Steady state kinetics of bisubstrate reactions. In:
Enzyme Kinetics, Chapter IX, pp. 560^610. Wiley, New York.
[39] Ganzhorn, A.J. and Plapp, B.V. (1988) Carboxyl groups near the
active site zinc contribute to catalysis in yeast alcohol dehydrogenase.
J. Biol. Chem. 263, 5446^5454.
[40] Amiguet, J.P. (1975) Wirkungsmechanismus der Hefe Alkoholdehydrogenase. Ph.D. thesis. Eidgeno«ssische Technische Hochschule, Zu«rich.
[41] Dickenson, C.J. and Dickinson, F.M. (1975) A study of the oxidation
of butan-1-ol and propan-2-ol by nicotinamide dinucleotide catalyzed
by yeast alcohol dehydrogenase. Biochem. J. 147, 541^547.
[42] Barshop, B.A., Wrenn, R.F. and Frieden, C. (1983) Analysis of numerical methods for computer simulation of kinetic processes: Development of KINSIM - A £exible, portable system. Anal. Biochem.
130, 134^145.
[43] Zimmerle, C.T. and Frieden, C. (1989) Analysis of progress curves by
simulation generated by numerical integration. Biochem. J. 258, 381^
387.
[44] Frieden, C. (1994) Analysis of kinetic data: practical application of
computer simulation and ¢tting programs. Methods Enzymol. 240,
311^322.
[45] Leskovac, V., Trivic¤, S. and Pericin, D. (2002) Isomerization of enzyme-coenzyme complexes in yeast alcohol dehydrogenase-catalyzed
reactions. Unpublished results.
[46] Northrop, D.B (2002) E¡ects of high pressure on enzymatic activity.
Biochim. Biophys. Acta 1595, 71^79.
[47] Eklund, H. and Bra«nde¤n, C.-I. (1987) Alcohol dehydrogenase. In:
Biological Macromolecules and Assemblies : Active Site of Enzymes
(Jurnak, F.A. and McPherson, A., Eds.), Vol. 3, pp. 73^142. Wiley,
New York.
[48] Chandra Sekhar, V. and Plapp, B.V. (1990) Rate constant for a
mechanism including intermediates in the interconversion of ternary
complexes by horse liver alcohol dehydrogenase. Biochemistry 29,
4289^4295.
[49] Cho, Y.K. and Northrop, D.B. (1999) E¡ects of pressure on the
kinetics of capture by yeast alcohol dehydrogenase. Biochemistry
38, 7470^7475.
493
[50] Leskovac, V., Trivic¤, S. and Latkovska, M. (1976) State and accessibility of zinc in yeast alcohol dehydrogenase. Biochem. J. 155, 155^
161.
[51] Havlis, J. and Studnickova, M. (1997) Zinc centers in alcohol dehydrogenase from horse liver and from baker’s yeast are metal dithiolenes. Bioelectrochem. Bioenerg. 43, 157^159.
[52] Magonet, E., Hayen, P., Delforge, D., Delaive, E. and Remacle, J.
(1992) Importance of the structural zinc atom for stability of yeast
alcohol dehydrogenase. Biochem. J. 287, 361^365.
[53] Jo«rnvall, H. (1977) Di¡erences between alcohol dehydrogenases.
Structural properties and evolutionary aspects. Eur. J. Biochem. 72,
443^452.
[54] Benetzen, J.L. and Hall, B.D. (1982) The primary structure of the
Saccharomyces cerevisiae gene for alcohol dehydrogenase I. J. Biol.
Chem. 257, 3018^3025.
[55] Russel, D.W., Smith, M., Williamson, V.M. and Young, E.T. (1983)
Nucleotide sequence of the yeast alcohol dehydrogenase II gene.
J. Biol. Chem. 258, 2674^2682.
[56] Ramaswamy, S., Kratzer, D.A., Hershey, A.D., Rogers, P.H., Arnone, A., Eklund, H. and Plapp, B.V. (1994) Crystallization and
preliminary crystallographic studies of Saccharomyces cerevisiae alcohol dehydrogenase I. J. Mol. Biol. 235, 777^779.
[57] Jo«rnvall, H., Eklund, H. and Bra«nde¤n, C.-I. (1978) Subunit conformation of yeast alcohol dehydrogenase. J. Biol. Chem. 253, 8414^
8419.
[58] Plapp, B.V., Ganzhorn, A.J., Gould, R.M., Green, D.W., Jacobi, T.,
Warth, E. and Kratzer D.A. (1990) Catalysis by yeast alcohol dehydrogenase. In: Enzymology and Molecular Biology of Carbonyl Metabolism 3 (Weiner, H., Wermuth, B. and Crabb, D.V., Eds.), pp.
241^251. Plenum Press, New York.
[59] Eklund, H., Plapp, B.V., Samama, J.-P. and Bra«nde¤n, C.-I. (1982)
Binding of substrate in a ternary complex of horse liver dehydrogenase. J. Biol. Chem. 257, 14349^14358.
[60] Cook, P.F. and Cleland, W.W. (1981) pH Variation of isotope e¡ects
in enzyme-catalyzed reactions. Biochemistry 20, 1797^1816.
[61] Plapp, B.V., Ganzhorn, A.J., Gould, R.M., Green, D.W. and Hershey, A.D. (1987) Structure and function in yeast alcohol dehydrogenases. In: Enzymology and Molecular Biology of Carbonyl Metabolism 3 (Weiner, H. and Flynn, T.G., Eds.), pp. 227^236, Alan R.
Liss, New York.
[62] Fan Fan and Plapp, B.V. (1999) Probing the a⁄nity and speci¢city of
yeast alcohol dehydrogenase I for coenzymes. Arch. Biochem. Biophys. 367, 240^249.
[63] Fan Fan, Lorenzen, J.A. and Plapp, B.V. (1991) An aspartate residue
in yeast alcohol dehydrogenase I determines the speci¢city for coenzyme. Biochemistry 30, 6397^6401.
[64] Weinhold, E.G., Glasfeld, A., Ellington, A.D. and Benner, S.A.
(1991) Structural determinants of stereospeci¢city in yeast alcohol
dehydrogenase. Proc. Natl. Acad. Sci. USA 88, 8420^8424.
[65] Trivic¤, S., Zeremski, J. and Leskovac, V. (1997) NADþ binding by
yeast alcohol dehydrogenase in the presence of pyrazole and a new
method for the determination of the enzyme active site concentrations. Biotechnol. Lett. 19, 809^811.
[66] Cleland, W.W. (1982) The use of pH studies to determine chemical
mechanism of enzyme-catalyzed reactions. Methods Enzymol. 87,
391^405.
[67] Nadolny, C. and Zundel, G. (1997) Fourier transform infrared spectroscopic stu-dies of proton transfer processes and the dissociation of
Zn2þ -bound water in alcohol dehydrogenase. Eur. J. Biochem. 247,
914^919.
[68] Fischer, H.F., Conn, E.E., Vennesland, B. and Westheimer, F.H.
(1953) The enzymatic transfer of hydrogens. 1. The reaction catalyzed
by alcohol dehydrogenase. J. Biol. Chem. 202, 687^697.
[69] Westheimer, F.H. (1987) Mechanism of action of pyridine nucleotides. In: Pyridine Nucleotide Coenzymes (Jurnak, F.A., McPherson,
A. and Avramovic, J., Eds.), Part A, pp. 253^322. Wiley, New York.
[70] Cook, P.F., Oppenheimer, N.J. and Cleland, W.W. (1981) Secondary
FEMSYR 1513 11-11-02
494
[71]
[72]
[73]
[74]
V. Leskovac et al. / FEMS Yeast Research 2 (2002) 481^494
deuterium and nitrogen-15 isotope e¡ects in enzyme-catalyzed reactions. Chemical mechanism of liver alcohol dehydrogenase. Biochemistry 20, 1817^1825.
Klinman, J.P. (1978) Kinetic isotope e¡ects in enzymology. In: Advances in Enzymology and Related Areas of Molecular Biology
(Meister, A., Ed.), Vol. 46, pp. 415^440. Wiley, New York.
Klinman, J.P. (1993) Hydrogen tunneling and coupled motion in
enzyme reactions. In: Enzyme Mechanism from Isotope E¡ects
(Cook, P.F., Ed.), pp. 127^147. Academic Press, New York.
Bahnson, B.J., Colby, T.D., Chin, J.K., Goldstein, B.M. and Klinman, J.P. (1997) A link between protein structure and enzyme catalyzed hydrogen tunneling. Proc. Natl. Acad. Sci. USA 94, 12797^
12802.
Cha, Y., Murray, C.J. and Klinman, J.P. (1989) Hydrogen tunneling
in enzyme reactions. Science 243, 1325^1330.
[75] Klinman, J.P. (1976) Isotope e¡ects and structure-reactivity correlations in the yeast alcohol dehydrogenase reaction. A study of the
enzyme-catalyzed oxidation of aromatic alcohols. Biochemistry 15,
2018^2026.
[76] Ramaswamy, S., Eklund, H. and Plapp, B.V. (1994) Structures of
horse liver alcohol dehydrogenase complexed with NADþ and substituted benzyl alcohols. Biochemistry 33, 5230^5237.
[77] Bahnson, B.J. and Klinman, J.P. (1995) Hydrogen tunneling in enzyme catalysis. Methods Enzymol. 349, 373^397.
[78] Kohen, A. and Klinman, J.P. (1999) Hydrogen tunneling in biology.
Chem. Biol. 6, R191^R198.
[79] Leskovac, V., Trivic¤, S. and Nikolic¤-DIoric¤, E. (1997) Temperature
dependence of the primary kinetic isotope e¡ect in hydride transfer
reactions with NADþ and NADH models. Bioorg. Chem. 25, 1^
10.
FEMSYR 1513 11-11-02