Essential role of arginine 235 in the substrate-binding of Lactobacillus plantarum D-lactate dehydrogenase.
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.
Department of Agricultural Chemistry, University of Tokyo.
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D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D003931:Diacetyl; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology*; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D005973:Glutamine; D006639:Histidine; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D003931:Diacetyl; D008239:Lysine