Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.
The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.
Karsten
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WE
Department of Chemistry, University of Akron, OH 44325-3601.
Viola
R E
RE
D001223:Aspartate-Semialdehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D005557:Formaldehyde / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010085:Oxidative Phosphorylation; D013379:Substrate Specificity
D005557:Formaldehyde; D001223:Aspartate-Semialdehyde Dehydrogenase; D005033:Ethylmaleimide