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Nucleophillic Enzymatic Reactions of Acetyl-CoA.

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Nucleophilic Enzymatic Reactions of Acetyl-CoA
By H . Eggerer[*l
Since acetyl-CoA does not form a carbanion[**l[l~]it was
difficult to determine the mechanism of enzymatic carboxymethylations of 0x0 acids by acetyl-CoA. An aldol-like
mechanism has now been demonstrated for two enzymes.
Three formal steps are necessary :
The reactants are bound to the enzyme in such a way that
their intramolecular conversion into the products follows
naturally. In the related enzyme catalyses and with citrate
synthase Mg2+ ions can be replaced by Lewis acids that
form part of the protein structure and can be arranged in
steric opposition to the group R of the a-ox0 acid. [VB 46 IE]
Lecture at Marburg (Germany) on November 18th. 1966
German version: Angew. Chem. 79, 383 (1967)
1) enolization of the acetyl-CoA, 2) addition of the enol to
the carbonyl group, giving the 2-hydroxyacyl-CoA, and
3) hydrolysis of this intermediate.
[ * ] Dr. H. Eggerer
Citrate synihase catalyses the synthesis of citrate from
acetyl-CoA and oxaloacetate. Steps 2) and 3) could be
demonstrated by simultaneous aldol fission and hydrolysis
by citryl-CoA, and the isolated step 3) by (S)-malyl-CoA.
Step 1) provides the key to understanding of the enzymatic
catalysis: step 1) requires the carboxylate anion of the
reactant oxaloacetate as basic co-factor [21. Its proof was
obtained with (S)-malate, which is bound by the enzyme in
place of oxaloacetate but cannot add to the enol. (S)-Malate
thus induces enol formation and leads to isotopic exchange
between the methyl hydrogen atom of the acetyl group and
tritiated water [ 3 J . Citrate that is synthesized in tritiated
water in the absence of (S)-malate is free from tritium;
acetyl-CoA is unlabeled after partial reaction with oxaloacetate, i. e. enolic acetyl-CoA obtained in the slowest step of
the synthesis reacts at once with the keto group, yielding
(S)-citryl-CoA. Since its “chemical hydrolysis” in neutral
aqueous solution occurs by way of 2-(carboxymethyl)-Zhydroxysuccinic anhydride [4,51, its enzymatic hydrolysis
presumably begins with the same step.
in the text.
[l] A . Markusu. B.Venneslund, J. biol.Chemistry233, 726 (1958).
[2] J. Bovi, 0. R . Martin, L. L. Ingraham, and P . K. Stumpf. _I
biol. Chemistry 234, 999 (1958).
[3] H. Eggerer, Biochem. Z. 343, 111 (1965).
[4] W. Buckel and H . Eggerer, Fall Meeting of the Gese/lsc/iuft
jiir physiologische Chemie, Marburg 1966. Abstr. p. 18.
[S] H. Eggerer, Liebigs Ann. Chem. 666, 192 (1963).
[6] G. H. Dixon, H . L. Kornberg, and P . Lund, Biochim. biophysics Acta 41, 217 (1960).
[?I H. Eggerer and A . Klerte, Europ. J. Biochem., in press.
[ 8 ] C . G. Swain and F. J . Brown, J. Amer. chem. SOC.74, 2538
Malate synthase catalyses the synthesis of malate from
acetyl-CoA and glyoxylate and is dependent on Mg2+ ions
as co-factors for this reaction [61. The enzyme enolises acetylCoA (step 1); for this step it is dependent on the Mg2L
ions f71, whose action is considered as acid-catalysis. The
thioester carbonyl group is polarized by linkage to the Lewis
acid, the methyl hydrogen atom becoming acidic.
Since the enolization is slow it seemed probable that for
each enzyme there are base- and acid-catalysis mechanisms
that act cooperatively with both enzyme systems. A chemical
model is Swain and Brown’s bifunctional catalyst 181. Accordingly a-0x0 acids induce enolization of acetyl-CoA by
malate-synthase; the efficiency of this induction increases
with decrease of their chain lenght i.e. with increasing
similarity to glyoxylic acid. Pyruvate inhibits the synthesis
of malate competitively (Ki= 10-3 M) and has measurable
affinity for the enzyme (KM = 10-3 M). The rate of enolization
in the “bifunctional enzyme system” and the rate of synthesis of malate are almost identical. Carboxylate anions and
Lewis acids act cooperatively: the enzymatic enolization
ceases if Mgz+ ions or pyruvate are removed. Since enolic
acetyl-CoA is formed in the slowest step of the synthesis
(proof as for citrate-synthase), it reacts at once with the
carbonyl group of the glyoxylate, giving (S)-malyl-CoA. If
used as a substrate, (S)-malyl-CoA is hydrolysed by
malate synthase (step 3).
The following reaction scheme is in accord with the experimental observations :
Institut fur Biochemie der Universitat
8 Miinchen 2 (Germany)
The acetyl-CoA carbanion and enol are not differentiated
Preparation and Reactions of Enamides
By F. Eiden‘*1
According to the conditions, reaction of substituted acetaldehydes with carboxamides, sulfonamides, or phosphoramides in the presence of acid catalysts yields enamides ( I ) ,
enamines (2), divinylamines (3), or diacylaminals ( 4 ) . If
hydroxamic acids are used in place of acid amides, then,
inter a h , N-hydroxyenamides [ ( I ) , R1 = OH], N-hydroxydivinylamines [(3), R1 = OH], or, again, enamides are
obtained; and use of hydrazides gives, inter a h , enehydrazides [ ( I ) , R1 = NHR].
+ H-N-C-R~
N-Hydroxyaminoalcohols, which can be formulated as
intermediates in the reactions of hydroxamic acids, can be
trapped in some cases as dihydro-l,4,2-dioxazoles(5) or
Citrate synthase: R
Malate synthase: R
Angew. Chem. internat. Edit. / Vol. 6 (1967) / No. 4
The enamides ( I ) are often obtainable in good yield and are
useful for synthesis of heterocyclic compounds. For instance :
4-hydroxyquinolines (7) result if in (2) X = CO-OAlkyl,
R1 = H, and RZ = Aryl; isoquinolines ( 8 ) if X = Aryl, RI =
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coa, nucleophillic, reaction, enzymatic, acetyl
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