The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation.
When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.
Craig
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Department of Biological Chemistry, University of Michigan, Ann Arbor.
Dekker
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D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000378:metabolism; D002104:Cadmium / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism / Q000494:pharmacology
D004220:Disulfides; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D002104:Cadmium; D009243:NAD; D013912:Threonine; D008345:Manganese; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D019807:Iodoacetic Acid