Inactivation of Escherichia coli L-threonine dehydrogenase by 2,3-butanedione. Evidence for a catalytically essential arginine residue.
Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics. The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase. Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e. 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection. Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues. In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+. Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site.
Epperly
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Department of Biological Chemistry, The University of Michigan, Ann Arbor 48109-0606.
Dekker
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D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000037:antagonists & inhibitors*; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002074:Butanones / Q000494:pharmacology*; D002384:Catalysis; D003512:Cyclohexanones / Q000494:pharmacology; D003931:Diacetyl / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D010422:Pentanones / Q000494:pharmacology; D010658:Phenylglyoxal / Q000494:pharmacology
D002074:Butanones; D003512:Cyclohexanones; D010422:Pentanones; D009243:NAD; C018031:1,2-cyclohexanedione; D001120:Arginine; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D003931:Diacetyl; C013186:2,3-pentanedione; D010658:Phenylglyoxal