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J Biochem. 1994 May;115(5):930-6.

Essential role of arginine 235 in the substrate-binding of Lactobacillus plantarum D-lactate dehydrogenase.

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1
Department of Agricultural Chemistry, University of Tokyo.

Abstract

Substitutions of the conserved Arg-235 with Lys and Gln induced drastic decreases in the catalytic efficiency of Lactobacillus plantarum D-lactate dehydrogenase (D-LDH). Both the mutant enzymes showed a marked resistance to 2,3-butanedione, by which the wild-type enzyme is rapidly inactivated unless NADH and oxamate are present. The pKa of the catalytic His was markedly shifted to the alkaline side by the Arg-to-Gln substitution, while it was not significantly shifted by the Arg to Lys substitution. The Arg-to-Lys replacement, by which the catalytic efficiency was less damaged, also induced decreases in kcat/Km for alternative substrates, such as 2-ketobutyrate, by approximately 10,000-fold, virtually the same level as in the case of pyruvate. Although both the wild-type and mutant enzymes exhibited lower kcat/Km for the alternative substrates than that for pyruvate, in the case of the mutant enzyme, the decrease in kcat/Km for the alternative substrates was mostly due to a decrease in kcat, while it was caused mainly by an increase in Km in the wild-type enzyme, suggesting that the mutant enzyme tends to form a nonproductive enzyme-substrate complex, in particular with more unfavorable substrates. The pH-dependence of the kinetic constants also indicated that there is a nonproductive binding that does not require the protonated or deprotonated form of the catalytic His residue. These results strongly suggest that Arg-235 plays an essential role in the tight and correct binding of substrate to the binding site of D-LDH, as in the case of Arg-171 in L-LDH.

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