Aspartate‐β‐semialdehyde dehydrogenase, from an Escherichia coli mutant derepressed for the biosynthesis of l‐lysine, has been purified to homogeneity. Its isoelectric point is pH 4.3. This enzyme has a molecular weight of 77000 and is composed of two identical or highly similar subunits of molecular weight 38000 ± 2000. Their N‐terminal amino‐acid sequence is Met‐Lys‐Asx‐Val‐Gly‐. Three cysteine residues per subunit were detected: two are reactive in the native enzyme and one is partially protected by the substrate. Formation of an acyl‐enzyme intermediate was also detected. Correlation of the 1H nuclear magnetic resonance spectrum of [4‐2H]NADPH produced from [4‐2H]NADP+ indicated that aspartate β‐semialdehyde dehydrogenase transfers the pro‐S hydrogen from NADPH (class B dehydrogenase). A short comparison with the corresponding yeast enzyme is given.

The substrate binding site of aspartate-P-semialdehyde dehydrogenase from Escherichia coli was studied by affinity labeling with ~-2-amino-4-oxo-5-chloropentanoic acid. The substrate analogue irreversibly inactivates the enzyme with pseudo-first-order kinetics and with a half-of-the-sites reactivity. The substrate aspartate P-semialdehyde protects the enzyme against the inactivation. A single group is labeled at the active site and is concluded to be the side-chain of a histidine residue. The amino acid sequence around the active site residue was established from a peptic digest of the labeled enzyme : Phe-Val-Gly-Gly-Asp-(modified residue)-Thr-Val-Ser.Different types of aldehyde : pyridine dinucleotide linked dehydrogenases have been isolated from different sources: e.g. glyceraldehyde-3-phosphate dehydrogenase of the glycolytic pathway, aspartate-P-semialdehyde dehydrogenase in the biosynthetic pathway of amino acids and unspecific aldehyde dehydrogenases in catabolic pathways. The relationships between these enzymes are unknown but may be further determined by structural studies [l]. In fact, the glyceraldehyde-3-phosphate dehydrogenases studied to date show high conservation in the sequence around the cysteine residue at the active site [2].We have studied aspartate-P-semialdehyde dehydrogenase, an enzyme which catalyses the phosphorylative oxidation of aspartate P-semialdehyde to aspartyl phosphate by NADP', in order to characterize its active site, and possibly to compare it with glyceraldehyde-3-phosphate dehydrogenase. These two enzymes catalyze the same type of reaction using the same kind of substrates, pyridine nucleotide coenzymes and inorganic phosphate. Both enzymes form intermediates with their respective substrates and both catalyze coenzyme reduction with the same stereochemistry [3]. The present study reports results from affinity labeling of the active site of aspartateAhhreviations. Tos-PheCHZCl, L-1-tosylamido-2-phenylethyl chloromethyl ketone; dansyl, 5-dimethylaminonaphthalene-1-sulphonyl.Enzymes. L-Aspartate-/?-semialdehyde : NADP

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