Abstract
γ-Cystathionases catalyze elimination of substituents from the β- or γ-positions of 4-carbon chain length amino acids with formation of α-ketobutyrate, but when N-ethylmaleimide is added to reaction mixtures the α-ketobutyrate is replaced by a product reflecting addition to the maleimide double bond of an intermediate with carbanion character at carbon 3. The product is α-keto-3-(3′-(N′-ethyl-2′,5′-dioxopyrrolidyl)) butyric acid, or KEDB. The reactive intermediate was postulated to be one of the three enamines: free aminocrotonate, aminocrotonate bound to the enzyme in an unprotonated form, or the Schiff base between aminocrotonate and the coenzyme, pyridoxal phosphate. The cystathionases have now been shown to catalyze stereospecific reactions with N-ethylmaleimide, yielding only one of the two possible diastereoisomers. The same isomer was formed by a third pyridoxal-P enzyme, cystathionine γ-synthase acting on O-succinylhomoserine, but a threonine dehydrase yielded the other isomer of KEDB from threonine. The last two enzymes differ from cystathionases in that the 3-carbon substrates, O-succinylserine and serine, respectively, do react in the presence of the maleimide to yield the analogue of KEDB less the methyl group, KEDP. This evidence that free enamino acids were not the intermediates that reacted with maleimide prompted an investigation of whether they were intermediates in any form. The basis for this study was that if they were, solvent tritium would be introduced into the β-position of newly formed pyruvate or α-ketobutyrate in a spontaneous step, and therefore the kinetic isotope effect would always be the same, regardless of which enzyme was used. Preliminary results suggest that the isotope effects may differ characteristically with different enzymes. A third study of the reactions of enamines with maleimides involved generating free enamines in the presence of the latter, through the action of hydrolytic enzymes on N-acyl derivatives of α-aminoacrylate and α-aminocrotonate. A kidney aminopeptidase produced KEDP from the first and a mixture of isomers of KEDB from the second. However, it is possible that the reactive species was an enzyme-bound enamine in this case also.
Highlights
Y-Cystathionases catalyze elimination of substituents from the /3- or y-positions of 4-carbon chain length amino acids with formation of cr-ketobutyrate, but when N-ethylmaleimide is added to reaction mixtures the Lu-ketobutyrate is replaced by a product reflecting addition to the maleimide double bond of an intermediate with carbanion character at carbon 3
The impetus for the present studies came from the discovery that y-cystathionase from Neurospora could form KEDB from p- as well as from y-substituted amino acids
In accord with earlier results (1) neither cystine nor lanthionine yielded any KEDP, a conclusion based until now on failure to find radioactivity in electropherograms in the area near KEDB. It became more certain with the availability of an authentic sample of KEDP, first isolated from a nonenzymic reaction mixture: and identified as described above
Summary
Y-Cystathionases catalyze elimination of substituents from the /3- or y-positions of 4-carbon chain length amino acids with formation of cr-ketobutyrate, but when N-ethylmaleimide is added to reaction mixtures the Lu-ketobutyrate is replaced by a product reflecting addition to the maleimide double bond of an intermediate with carbanion character at carbon 3. The last two enzymes differ from cystathionasesin that the d-carbon substrates, 0-succinylserine and serine, respectively, do react in the presenceof the maleimide to yield the analogueof KEDB lessthe methyl group, KEDP This evidence that free enamino acids were not the intermediates that reacted with maleimide prompted an investigation of whether they were intermediates in any form. As mentioned in a preliminary report (5), when substrates were discovered which were P-substituted but of 4-carbon chain length (Fig. 2, V), it was found that they did yield KEDB
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