The Reaction of β‐Chloroglutamic Acid with Glutamate‐Aspartate Transaminase

Abstract

Porcine glutamate-aspartate transaminase catalyzes a @-elimination reaction with both the threo-and erythro-isomers of 8-chloroglutamate ; chloride, ammonia, and a-ketoglutarate are formed in equimolar amounts. The latter product was characterized as the 2,4-dinitrophenylhydrazone and by catalytic hydrogenation of this derivative to glutamic acid.The 8-elimination reaction is catalyzed by highly purified glutamate-aspartate transaminase ; the reaction is not catalyzed by the phosphopyridoxamine form of the enzyme, the apoenzyme, or by free pyridoxal 5'-phosphate. There is no detectable transamination between B-chloroglutamate and either oxaloacetate or a-ketoglutarate. Incubation of either the holoenzyme or the apoenzyme with p-chloroglutamate does not result in any detectable loss of enzyme activity. I n the presence of N-ethylmaleimide much less a-ketoglutarate than ammonia is formed; this is consistent with the idea that a reactive carbanion intermediate reacts with N-ethylmaleimide.The novel 8-elimination reaction with glutamate-aspartate transaminase demonstrated here supports the idea that in some instances the specificity of an enzymatic reaction can be determined by the structure of the substrate.Glutamate-aspartate transaminase catalyzes the reversible transfer of an amino group fromL-aspartate to a-ketoglutarate forming oxaloacetate and L-glutamate; other amino acids can also undergo transamination with this enzyme although they are less efficient substrates than the glutamate-aspartate pair [I]. There has been considerable interest in the reaction of substrate analogues with glutamate-aspartate transaminase in an effort to elucidate some of the intermediate steps of the reaction. Thus, in a study of the reaction between the transaminase and a-methylaspartate (which lacks an a-hydrogen atom and hence cannot undergo transamination) two new absorption bands were observed, one at 430 mp and the other at 360 mp [2]. It was proposed that these are due to coenzyme-substrate aldimine derivatives analogous to those formed with the glutamateaspartate substrate pair before the loss of the a-hydrogen atom. When erythro-/3-hydroxyaspartic acid is incubated with glutamate-aspartate transaminase there is a dramatic increase in the absorption a t 490 mp; this is followed by a slow increase in the absorption a t 330 mp (attributed to the phosphopyridoxamine enzyme) and a corresponding formation Enzymes. Glutamate-aspartate transaminase, or L-aspartate: 2-oxoglutarate aminotransferase (EC 2.6.1.1) ; glutamic dehydrogenase, or L-glutamate : NAD oxidoreductase (deaminating) (EC 1.4.1.2) ; malic dehydrogenase, or L-malate: NAD oxidoreductase (EC 1.1.1.37); glutamic decarboxylase, or L-glutamate: 1-carboxy-lyse (EC 4.1.1.15).18 European J. Biochem., Vol. 5 of keto acid. However, little, if any, of the intcrmediate absorbing a t 490mp is detected when threo-@-hydroxyaspartate is the substrate. It has been suggested that the species absorbing a t 490 mp is a semiquinoid coenzyme-substrate derivative [3]. The reason for the d...

Inactivation of Brain and Kidney Aspartate Aminotransferases by <i>S</i>-(1,2-Dichlorovinyl)-<i>L</i>-Cysteine and by <i>S</i>-(1,1,2,2-Tetrafluoroethyl)-<i>L</i>-Cysteine

“…For example, the enzyme catalyzes elimination of chloride from tlireoand eov/tro-p-chloro-DL-glutamates; the enzyme is not inactivated by these compounds presumably because the product is not aminoacrylate [32], The enzyme also cat alyzes a P-elimination reaction with p-chloroalanine [33,34] and with L-serine-O-sulfate [35]. However, in these cases the process is associated with inactivation of the Inactivation of Aspartate Aminotransferase by Cysteine .S'-Conjugaics enzyme.…”

“…This 'preference' for a p-eliminatioti reaction is similar to that observed with p-chloroglutamate. Pig heart cytAspAT catalyzes an exclusive elimination reaction rather than a transaminase reaction with this compound [32], Apparently, for both DCVC and [3-chlorogIulamate the type of reaction catalyzed by AspAT is dictated by structure of substrate rather than by a built-in function of the enzyme fcf 32].…”

Inhibition of Bacterial Growth by β-Chloro-D-Alanine

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i3-Chloroamino acids have been shown recently to bind efficiently to transaminases and decarboxylases and to undergo #3-elimination reactions in situ (7,8). In some cases the enzyme catalyzing the (3-elimination reaction is irreversibly inactivated presumably by reaction of the enzyme-bound aminoacrylic acid with some functional group of the protein (8).

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Kinetic study of the interaction between aspartate aminotransferase and threo-.beta.-chloroglutamate

“…These fast optical density changes are followed by slower ones associated with the reappearance of the spectrum of the free enzyme as substrate is exhausted. The kinetics of the fast process can be accommodated in a simple scheme, reflecting the formation of an enzyme-substrate complex :The following values for the kinetic constants have been obtained: kl = 7 X lo2 M-' sec-' and k-' = 7.5 sec-The apparent equilibrium constant can be determined from the amplitude of the spectral change related to the fast process as a function of substrate concentration; the I t has been recently shown (Manning et al, 1968) that a substrate analog containing a strongly electronegative group in the P position, threo-P-chloroglutamate, undergoes, in the presence of catalytic amounts of aspartate aminotransferase,' a @-elimination reaction, with release of C1-, NHa+, and a-ketoglutarate. There is already quite strong evidence that this p-elimination reaction is specifically catalyzed by the aminotransferase itself, and not by some impurity (Manning et al, 1968).…”