Download R-C-SCoA (acyl CoA) O

VII. Some More Cofactors and the Pyruvate Dehydrogenase Complex.
Coenzyme A: A Cofactor that uses the SH group.
CoA consists of phosphopantetheine coupled to AMP via a phosphodiester bond. The symbolic
representation is CoA or CoA-SH. CoA is a reagent that activates -COOH groups for reaction at neutral pH. This is
achieved by formation of the acyl thioester
O
R-C-SCoA
(acyl CoA)
in a reaction that utilizes ATP and is catalyzed by enzymes called acyl CoA synthetases; acyl AMP is a catalytic
intermediate. Acetyl CoA synthetase a.k.a. "acetate activating enzyme" or ACETATE THIOKINASE is a specific
example:
acetate + ATP + CoA ⇔ Acetyl-CoA + AMP +PPi
∆Go' = - 0.6 kcal/mole
PPi ⇔ 2Pi
∆Go' = - 5.3 kcal/mole
VII-1
The net change in ∆Go' = - 5.9 kcal/mole so synthesis of acetyl CoA is essentially complete.
The identity of the products–in this case AMP plus Ppi-has a significance. The carboxyl group of an
organic acid (e.g. acetate) is an unactivated, low-energy, resonance-stabilized anion that is not easily attacked by a
nucleophile (e.g. CoAS-); it is extremely difficult to remove one of the two carboxylate oxygen atoms in a
displacement reaction. or this to occur the carboxylate group must be activated and a good leaving group introduced
(alternatively said, the poor leaving group OH must be replaced by a good leaving group). When ATP is used to
activate the carboxyl function either of two possible leaving groups can be introduced--AMP or P. When the
products of the reaction are ADP and Pi we have evidence for the intermediate formation of carboxyl phosphate;
when the products are AMP and PP then we infer that the -COOH is adenylated. (Both -P and -AMP are good
leaving groups--they are the conjugate base to a strong acid.)
We might speculate that the individual steps of the thiokinase reaction are:
Enz + acetate + ATP ⇔ acetyl-AMP~Enz + PPi
acetyl-AMP~Enz + CoA ⇔ acetyl CoA + AMP + Enz
Such reactions are called displacement reactions because the enzyme displaces a portion of one (or more) of the
substrates. The verification of presumed displacement mechanisms such as this one relies upon
The Isotope Exchange Technique
For a substitution/displacement mechanism of type just described there is a very powerful technique for
verifying the mechanism of the reaction. Consider the simple case:
A + Enz ⇔ A.Enz ⇔ P + Enz
If we take some non-radioactive A and incubate it with the enzyme then the conversion of A to P will occur
and soon we will have A and P present at their equilibrium concentrations. There will be no additional net
conversion of A to P or of P back to A. These two processes will be proceeding at equal rates. If, at this point, we
add some radioactive A to the reaction mixture radioactivity would begin to appear in P and the relative proportions
of isotope in A and P will eventually achieve the equilibrium ratio of A and P.
A more interesting situation arises with more complex enzymes. The classic example is sucrose
phosphorylase.
Sucrose + P ⇔ Glucose-1-P + Fructose
Representing sucrose as G-F the postulated mechanism is:
G-F + Enz ⇔ Enz~G + F
---- (a)
Enz~G + P ⇔ Enz + G-1-P
---- (b)
i.e. the reaction proceeds with the intermediate formation of an enzyme-glucose covalent complex (Enz~G) that is
unable to dissociate to yield free glucose and enzyme.
If we take enzyme and add G-F the equilibrium depicted in reaction (a) will proceed but the second step,
which requires P, cannot occur. On adding radioactive fructose (*F) to the reaction mixture, this will mix with the F
produced by the forward reaction (a) and, upon reversal of this reaction, some *F will appear in G-F i.e. some G-*F
is formed. On the other hand, addition of *G to the mixture does not lead to the formation of *G-F. This observation
implies that when sucrose is added to sucrose phosphorylase the fructose is split out into the medium while the
glucose is retained in the enzyme.
Half-reaction (b) can be examined by incubating the enzyme with G-1-P in the absence of F. In this case it
is found that added 32P can be incorporated into the G-1-P but added *G does not. This establishes that when G-1-P
is added to the enzyme P is split out and the G once more retained. These observations are required by steps (a) and
(b).
VII-2
Exchange Data for acetate activating enzyme (thiokinase):
Exchange Studied
Also Required
Not req. to demonstrate the exchange
1.
AMP ⇔ ATP
Acetate, CoA
----
2.
PP ⇔ ATP
Acetate
CoA
3.
Acetate ⇔ AcCoA
AMP, PP
CoA
Exchange # 1 is read "exchange labeled AMP into ATP" (note that it's always the smaller part into the bigger part).
For exchange #1 to occur we must have present ATP, acetate and CoA (not AMP as it is formed by the first reaction
on p. VII-2). Persuade yourself that these are consistent with the mechanism given above. In exchange #1 the first
species i.e. AMP, will be present in vanishingly small amounts (detectable only because of its intense radioactivity)
while the second species (ATP) is present in finite amounts. Thus is contemplating this scenario you should pretend
that the [AMP] is essentially zero.
Acyl derivatives of CoA undergo a variety of metabolic reactions that may be classified into 4
groups of which the first two are the most important.
TYPE 1 or HEAD ACTIVATION (enhances electrophilicity of carbonyl C)
These are reactions involving attack of nucleophilic reagents at the acyl (carbonyl) carbon atom, with
transfer of the acyl function to the attacking reagent and release of CoA. This mode of reaction is called HEAD
activation because the end of the acyl function nearest to the CoA becomes attached to the nucleophile. It can be
represented generally as:
O
O
R
C
S
CoA
R
C Nuc + CoA S-
..Nuc
(CoA is expelled)
where Nuc, the attacking nucleophile, is varied e.g. phosphate, amine, water, alcohol, thiols, hydride, or a carbanion.
Note that this reaction occurs more readily than that of the unactivated acid because (i) the C=O is electrically neutral
compared to the carboxylate and (ii) the CoAS- is a much better leaving group than is OH-.
Specific Examples are:
a) Nuc = phosphate: Succinyl CoA synthetase
succinyl CoA + Pi + GDP ⇔ succinate + CoA + GTP
b) Nuc = Amine: Glucosamine Acyl Transferase
acetyl CoA + glucosamine ⇔ N-acetyl glucosamine + CoA
c) Nuc = Water: Acetyl-CoA hydrolase
acetyl CoA + H 2O ⇔ acetate + CoA
d) Nuc = Alcohol: glycerophosphate acetyl transferase
2 fatty acyl CoA + L-a-glycerophosphate ⇔ diglyceride-P + 2 CoA
e) Nuc = Thiol: Lipoic Transacetylase
acetyl CoA + dihydrolipoate ⇔ CoA + S-acetyl lipoate
f) Nuc = Hydride: Acyl CoA Reductases.
acetyl CoA + NADH + H+ ⇔ acetaldehyde + CoA + NAD
g) Nuc = Carbanion: β-ketothiolase
VII-3
acetoacetyl CoA + CoA ⇔ 2 acetyl CoA
TYPE 2 or TAIL ACTIVATION (Enhances nucleophilicity of α-CH2).
These are reactions involving condensation of the alkyl carbon of the acyl-CoA presumably by activation of
the alkyl carbon by formation of its carbanion. This is called TAIL activation because the target reagent is attached
to the acyl function by the end furthest from the CoA.
COO-
O
-O
C
CH3CHCOSCoA
OH
..
CH3CHCOSCoA
In this reaction the carbanion on the α-C of propionyl CoA attacks the bicarbonate to make methylmalonyl
CoA. The facile character of this reaction is attributed to the increased acidity of the thioester compared to the oxoester. Thioesters are 100-1000 times more acid which means that they have a much greater tendency to undergo
proton dissociation at the methylene function immediately adjacent to the sulfur; the developing negative charge that
is produced by this dissociation is stabilized by delocalisation over the C=O giving partial enolate character to the
intermediate, and by the polarizability of the sulfur. An excellent example is citrate synthetase which we will
consider in Ch. 8.
We also have the "Siamese Twin" reaction:
2 acetyl CoA ⇒ acetoacetyl CoA
in which one acetyl CoA undergoes HEAD and the other undergoes TAIL activation (this causes confusion in the
classification).
The next two types are for completeness only; they will not be required.
TYPE 3
Reactions involving additions to derivatives of CoA species that have an olefinic link in the acyl moiety
e.g. the reaction catalyzed by Enoyl-CoA Hydratase.
H2O + RCH=CH-CO-S-CoA ⇒ RCH 2-CH(OH)-CO-S-CoA
TYPE 4
Reactions involving acyl group interchange. e.g. acetoacetic-CoA transferase:
acetoacetyl CoA + CoA ⇒ 2 acetyl CoA
VII-4
OTHER SH COENZYMES: LIPOIC ACID, ACYL CARRIER PROTEINS AND COENZYME M.
1: Lipoic acid is 6,8-dithiooctanoic acid. It is a cyclic disulfide:
COOH
COOH
s
s
2e, 2H +
s
H
s
H
Peptide Bond with lysine
Lipoic acid undergoes facile oxidation-reduction in a 2-electron process that converts the disulfide to two
thiol functions. Eo' = -0.28 volts and changes with pH a slope of -0.06 volts per pH unit. WHY? The value of 0.28 v is very low for SH-SS potentials and is a result of the formation of the very stable 5-membered ring; the ring
formation is the driving force for losing the pair of electrons. The reduced form is called dihydrolipoic acid.
Lipoic acid is commonly attached to proteins via a covalent bond between the epsilon-NH2 of a lysine
function and the -COOH group that is present on the sidearm of the lipoic acid. As this is an amide bond the bound
form of lipoic acid is called lipoamide.
2: Acyl Carrier Protein. An analog of CoA in which the 4'-phosphopantetheine is linked to the –OH of a
serine residue in a protein (as opposed to the phosphate of AMP). You will learn more about this protein when
considering fatty acid synthesis.
3: (Ignore this for Bios 302). Coenzyme M: very primitive, found in the methanobacteria. The structure is
HSCH 2CH2SO3. It participates in thiol-disulfide redox reactions. In addition it can act as an analog for B12 and can
carry one-carbon fragments: (Walsh, 864).
S
S
SO32e
SO3-
C
SO3CoM-
o-
o
CoM
2S
6e CH CoM
CoM C
3
O
O
In methanogens methylCoM (CH 3-SCoM) reacts with a RSH to release CH4 and make RS-SCoM. These bugs make
400 million tons of methane each year
VII-5
THIAMINE PYROPHOSPHATE
Thiamine pyrophosphate is the biochemically active form of thiamine, vitamin B1. It consists of a
thiazolium ring, the functional component, plus a pyrimidine nucleus connected together by a methylene bridge. The
pyrophosphate is absent in the vitamin. Because this vitamin is neither synthesized nor stored it must be continually
provided in the diet (polished rice ⇒ beri-beri). The nitrogen of the thiazolium ring is quaternized, and this is a key
aspect in the function of this molecule. NMR has shown that the H atom on C2 of the thiazolium ring is very
labile, it is very acidic (pK is about 12). The C2 carbanion formed by removal of the H+ is stabilized by the
favorable coulombic interaction with the adjacent cationic nitrogen. This dipolar ion is called an ylid (pronounced
ilid), which is defined as a species in which two adjacent atoms have formal opposite charges but the negative atom
cannot donate its unshared pair to the bond between them since the positive atom already has a filled octet. The
im portant property of this species is the facile reaction of the lone pair on C2 to add to carbonyl groups of -keto
acids or -keto alcohols (i.e. sugars).
VII-6
Addition to α-keto-acid (e.g. pyruvate).
Addition to α-keto alcohol (e.g. fructose)
Each of the reactions of TPP enzymes begin with reaction of the ylid with the carbonyl group of
the appropriate keto substrate followed by C-C bond cleavage and production of an "active aldehyde" - an aldehyde
moiety attached to C2 of TPP. The structure of the active aldehyde will be dictated by the specific substrate but
formally it can be represented as in HETPP but with the -CH3 replaced with -R.
VII-7
Reactions of HETPP
(i) Transfer to an electrophile (typically a proton or another carbonyl compound) e.g. pyruvate decarboxylase.
(ii) Transfer to an oxidizing agent. (the pyruvate dehydrogenase complex).
VII-8
Reactions of Glycolyl TPP
(1) Addition to an aldo-sugar of length n to yield keto-sugar of length n+2
e.g. transketolase
CH 2OH
CHO
O
CH2 OP
CH2OP
Sedoheptulose-7-P
+
Erythrose-4-P
Ribose-5-P
+
F-6-P
(ii) Acetyl formation. Loss of water and prototropic shift ⇒ internal oxidation e.g. phosphoketolase.
VII-9
Net reaction:
F-6-P + Pi ⇒ erythrose-4-P + acetyl P (high energy).
(Note that some texts (but not V&V) use hydroxyethylideneTPP rather than hydroxyethylTPP; the abbreviation
HETPP is unchanged).
VII-10
FLAVINS
The flavin prosthetic groups are the biochemically important form of RIBOFLAVIN, the water-soluble
vitamin B2.
CH2 OH
H C
OH
H C
OH
H C
OH
CH2
H3 C
H3 C
N
N
O
NH
N
O
Two functional flavins occur commonly. The simplest is Flavin Mononucleotide (FMN) which is obtained
by condensation of N10 of the isoalloxazine ring with the 5-C sugar alcohol RIBITOL; note that this is not a
glycosidic bond because ribitol does not have a -C=O and thus cannot have a hemi-acetal. It is always in the open
chain form; thus this is "not quite a nucleotide". In FMN the ribitol is phosphorylated at C5.
CH2O-P-P-ribose-Adenine
H C
OH
H C
OH
H C
OH
CH2
H3 C
H3 C
N
N
O
NH
N
O
The more complicated and more abundant flavin is called Flavin Adenine Dinucleotide (FAD). It is a
dinucleotide obtained by the formation of a phosphodiester bond between the phosphate of FMN and the phosphate
of AMP. It is analogous to NAD except that the nicotinamide has been replaced by isoalloxazine and the ribose of
NMN by ribitol.
When we focus on the chemical reactivity of the flavin we simply draw the three-ring isoalloxazine portion
and represent the remainder of the dinucleotide by R-. The most important chemical reaction of flavin is a reversible
oxidation reduction; this can be written as a 1,5 addition of 2H to give dihydroflavin. Formally this reaction
resembles the addition of 2H to quinone to give hydroquinone:
VII-11
In contrast to NAD+ which is a mandatory 2-e acceptor, flavins can accept electrons either one or two at a
time; in the former case there is the intermediate formation of the 1-e reduced semiquinone or free radical form. The
existence of such species in enzyme reactions has been demonstrated numerous times using electron paramagnetic
resonance (epr).
1) Redox Potentials. Typical values are:
F + e + H+
FH + e + H+
Mean: F + 2e + 2H+
⇔
⇔
FH
FH2
E1 = - 0.24 v.
E2 = - 0.18 v.
⇔
FH2
Eo' = - 0.21 v.
Just as we saw for NADH, when bound to a protein these values can be significantly changed. For example
in one flavoenzyme (called old yellow enzyme) Eo' = - 0.12v, while in a second protein (called electron transfer
protein or flavodoxin) E1 = - 0.11 v. while E2 = - 0.371 v. i.e. Eo' = - 0.24 v.
VII-12
Other properties of Flavins.
Optical and Fluorescence Spectra.
1.0
_______ Oxidized Flavin absorbance
.............Reduced
Flavin absorbance
0.8
- - - - - - -Oxidized Flavin fluorescence
0.6
0.4
0.2
0.0
350
400
450
500
550
600
Wavelength
These are very similar for all the derivatives and are basically the spectra of the parent isoalloxazine ring
system.
The oxidized species is lemon-yellow and emits a green fluorescence. The fully reduced flavin is essentially
colorless and does not exhibit (significant) fluorescence. These characteristics are inverted relative to NAD+ which is
colored and fluorescent when in the reduced state. The semiquinone or half-reduced flavins have new absorption bands
to longer wavelength and are either blue or red to the eye depending upon the whether-or-not N5 is protonated (blue)
or deprotonated (red).
When embedded in an enzyme these characteristic colors are essentially unaltered and the color changes
which take place when a flavoenzyme is oxidized or reduced is an important experimental observable in studying the
mechanisms of these enzymes.
3) Binding to Proteins
Most commonly flavins are attached to the their respective protein by non-covalent interactions. Typically
the flavin is found lying in a cleft in the protein's surface and makes multiple H-bonding, hydrophobic and ionic
contacts with the polypeptide in much the same way as NAD + is attached to its respective enzyme. Normally the
flavin is liberated on denaturation of the protein component.
VII-13
However in some flavoproteins the flavin is attached covalently. Usually this is a result of bond formation
between an amino acid and C-8 of the isoalloxazine ring. Frequently histidine is the participating amino acid. An
important example of this is provided by the enzyme succinic dehydrogenase (SDH) which we will be considering
later. On acid hydrolysis of SDH 8a-histidyl FAD is obtained.
As with NAD, FAD can form a hairpin configuration in solution, and dynamically interconvert between
open and hairpin forms. However in those FAD flavoproteins for which X-ray structures are available the FAD is
bound in an extended manner, just as we saw for the binding of NAD to the appropriate dehydrogenase.
Modes of Reactions of Flavoproteins.
Because of the alternative modes of reduction three are 3 broad groups of reaction:
1) Flavoprotein oxidases in which the flavin receives and donates electrons two at a time e.g. amino acid
oxidases.
Alanine + E-FAD ⇔ E-FADH2 + imino acid
E-FADH2 + O2 ⇔ E-FAD + H2O2 (toxic)
The imino acid hydrolyzes spontaneously to pyruvate releasing ammonia..
2) Flavoprotein transformases: (2e ⇔ 1e)
a) Ferredoxin-NAD+ oxidoreductase: (photosynthesis, 2 x 1e --> 2e)
Fd- + E-FAD + H+ ⇔ Fd + E-FADH (Fd denotes ferredoxin, a 1e donor)
Fd- + E-FADH + H+ ⇔ Fd + E-FADH 2
E-FADH2 + NADP+
⇔ E-FAD + NADPH + H+
b) NADH-cytochrome b5 oxidoreductase: (microsomes, 2e --> 2 x 1e)
NADH + H+ + E-FAD ⇔ NAD+ + FADH2
E-FADH2 + b +3 ⇔ E-FADH + b+2 + H + (b denotes cyt. b5 , a 1e acceptor)
E-FADH + b +3 ⇔ E-FAD + b+2 + H +
Note that 2a and 2b are not basically different except that they are written in the direction of spontaneous
change.
VII-14
3) Flavin working exclusively as a 1-electron reactant:
FMN + e + H+ ⇔ FMNH
Flavodoxin
FMNH + e + H+ ⇔ FMNH 2
No example known to me.
But in addition to 1-3 there is one not-so-obvious family of reactions, the so-called mixed function oxidases
which serve to hydroxylate organic, frequently aromatic, compounds (e.g. phenol to catechol)
The generic reaction is:
AH + NADH + H+ + O2 ----> AOH + NAD+ + H2O.
where AH is the substrate. Note that dioxygen is split and one atom of oxygen is reduced to water and the other
incorporated into the organic substrate.
Mixed function oxidases are important in biosynthetic reactions.
VII-15
The Pyruvate Dehydrogenase Complex: The Entry to the TCA Cycle
We turn now to those decarboxylations of alpha-keto acids that proceed with simultaneous oxidations. Such
reactions are among the most important reactions of metabolism; indeed the very next reaction we will study–the
preparation of pyruvate for processing by the citric acid cycle–is a prime example. In this case the responsible
enzyme is called the Pyruvate Dehydrogenase Complex (PDC); it is a member of a group of enzymes called the
alpha-keto acid dehydrogenases. These dehydrogenases are multi-subunit multi-enzyme complexes. The best
characterized preparation of PDC is that obtained from E. coli. This enzyme consists of 60 polypeptides comprising
3 different subunits. These are divided up into three enzymes (E1, E2 and E3) as follows:
E 1p: 12 x 2: 12 subunits of pyruvate dehydrogenase, a TPP dependent oxidative decarboxylase (consequently this
subunit is sometimes referred to as pyruvate decarboxylase). Each subunit contains 2 chains (αβ) each of mass = 92
kDa. There is 1 TPP per α subunit.
E 2p: 8 x 3: 8 subunits of a transacetylase with 3 chains/subunit, each chain of mass = 65 kDa and 2 lipoic
acids/chain. In the mammalian enzyme this core contains 20 subunits.
E 3p: 6 x 2: 6 subunits of a FAD dependent Dihydrolipoyl dehydrogenase with 2 chains/subunit, each of mass = 56
kDa. There is 1 FAD per chain.
What is the total MW? 4.5 million!
VII-16
These 42 subunits are arranged in a very regular structure that approximates a cube. The core of the cube
consists of the 8 subunits of the transacetylase (E2). The 12 subunits of the pyruvate dehydrogenase lie one on each
of the 12 edges of the cube; the 6 subunits of the flavoprotein lie one on each of the 6 faces of the cube.
The Reaction Sequence.
The first step in the catalytic sequence is the reaction of pyruvate with the pyruvate dehydrogenase with the
formation of the activated C2 fragment HETPP and the release of CO2. This reaction is the same as that we wrote
earlier for pyruvate decarboxylase. However the present enzyme does not convert HETPP to acetaldehyde and TPP.
Pyruvate + TPP- ⇒ HETPP - + CO2
E 1α
(Occurs on edge, α subunit of E1)
The second reaction in the sequence is an acyl transfer reaction catalyzed by dihydrolipoamide transacetylase.
In this enzyme each subunit contains the prosthetic group lipoic acid covalently attached to a lysine residue in the
polypeptide by means of an amide bond. Thus the functional dithiolane ring is attached to the end of a relatively
long, flexible chain that provides an arm about 14Å long for possible motion. The disulfide bond of the ring is
thought to be attacked by the HETPP- carbanion generating a thiohemiacetal as was explained in the lecture on TPP:
HETPP - + lipS 2 ⇒ acetyl lipoate + TPPE1α
E2
E 1β
E2
E 1α
(occurs in core)
This reaction is similar to that we studied with triose phosphate dehydrogenase. The sp3 carbon of the
hemiacetal of HETPP is thus converted to the sp2 carbon in the acetyl-thioester link of an acetyl lipoamide. Now the
product is at the final oxidation state and is a thioester. The next step is acyl transfer from the thiol of lipoamide to
the thiol of CoA i.e. a trans-thiolation.
⇒ CH 3 CO SCoA + Lip(SH)2
E2
E2
At this point the overall conversion of the substrate keto-acid to the product, acyl CoA, has been completed
but the lipoamide prosthetic group has been left in the 2-e reduced state, the 2-e originating from the oxidation of the
VII-17
pyruvate. In this dihydro-form the lipoate is unable to effect a second turnover and it is the job of the third enzyme,
the dehydrogenase, to reoxidize the dihydrolipoamide back to the disulfide form. It is speculated that with its
swinging arm of 14 Å, the lipoyl moiety could swing up to 28Å, from the initial site of acylation (E1α), to the
flavin prosthetic group which will implement the reoxidation reaction. The dithiol reduces the bound FAD to
FADH2 and is itself re-oxidized back to the disulfide.
lip(SH)2 + FAD ⇒ lipS2 + FADH2
E2
E3 E 3 E 2
E3
(FACE)
One last task remains before the catalytic cycle is complete! The FADH2 must be reoxidized. This is
accomplished by NAD+.
FADH + NAD+ ⇒ FAD + NADH + H+
2
E3
E3 E3
The overall stoichiometry for the pyruvate dehydrogenase reaction is therefore:
CoA + Pyruvate + NAD+ ⇔ acetyl-CoA + NADH + CO2 + H+
In this reaction the prosthetic groups TPP, lipoamide and FAD function catalytically; the product, acetylCoA, will function as a substrate for a subsequent enzyme, particularly citrate synthase (a coming attraction).
Note that the mammalian PDC (but not the E. Coli enzyme) is under metabolic regulation. When a source
of energy is abundant e.g. [ATP] is high
E-OH + ATP
⇒
E 1-kinase
ACTIVE form
E-P
E-P + ADP
INACTIVE form
phospho-E1 phosphatase
⇒
E-OH + P
Phosphorylation modifies the α-chain of E1 by reaction at a serine residue. The kinase and phosphatase are bound to
the dehydrogenase (E1) component of the complex and are thus poised to modify the complex in response to
metabolic demand (compare with PFK).
(The structural properties of the mammalian enzyme have been summarized recently (FASEB J. Nov. 1990)
while the X-ray structure of the enzyme from the bacterium Azotobacter vinelandii has been determined to atomic
resolution (Science Vol. 255, -1544 (1992)).
VII-18