Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.
The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.
Kochhar
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Nestlé Research Centre, Vers-chez-les-Blanc, Lausanne, Switzerland.
Hunziker
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Leong-Morgenthaler
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Hottinger
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H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013379:Substrate Specificity
D000596:Amino Acids; D011773:Pyruvates; D009243:NAD; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase