Identification of an essential cysteine in the reaction catalyzed by aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.
The enzyme L-aspartate-beta-semialdehyde dehydrogenase from Escherichia coli has been studied by oligonucleotide-directed mutagenesis. The focus of this investigation was to examine the role of a cysteine residue that had been previously identified by chemical modification with an active site directed reagent (Biellmann et al. (1980) Eur. J. Biochem. 104, 59-64). Substitution of this cysteine at position 135 with an alanine results in complete loss of enzyme activity. However, changing this cysteine to a serine yields a mutant enzyme with a maximum velocity that is 0.3% that of the native enzyme. This C135S mutant has retained essentially the same affinity for substrates as the native enzyme, and the same overall conformation as reflected in identical behavior on gel electrophoresis and in identical fluorescence spectra. The pH profile of the native enzyme shows a loss in catalytic activity upon protonation of a group with a pKa value of 7.7. The same activity loss is observed at this pH with the serine-135 mutant, despite the differences in the pKa values for a cysteine sulfhydryl and a serine hydroxyl group that have been measured in model compounds. This observed pKa value may reflect the protonation of an auxiliary catalyst that enhances the reactivity of the active site cysteine nucleophile in the native aspartate-beta-semialdehyde dehydrogenase.
Karsten
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Department of Chemistry, University of Akron, OH 44325-3601.
Viola
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D000595:Amino Acid Sequence; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D015183:Restriction Mapping
D009838:Oligodeoxyribonucleotides; D001223:Aspartate-Semialdehyde Dehydrogenase; D003545:Cysteine