Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity.
The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.
Angeles
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Department of Chemistry, University of Akron, Ohio 44325-3601.
Smanik
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Borders
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Viola
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D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001222:Aspartate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006715:Homoserine Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D010770:Phosphotransferases / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology; D014466:Ultraviolet Rays
D007093:Imidazoles; D000429:Alcohol Oxidoreductases; D006715:Homoserine Dehydrogenase; D010770:Phosphotransferases; D001222:Aspartate Kinase; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate; C012160:N-acetylimidazole