Glutamate 264 modulates the pH dependence of the NAD(+)-dependent D-lactate dehydrogenase.
Recently, we amplified the Lactobacillus bulgaricus NAD(+)-dependent D-lactate dehydrogenase gene by the polymerase chain reaction, cloned and overexpressed it in Escherichia coli (Kochhar, S., Chuard, N., and Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 185, 705-712). Polymerase chain reaction-amplified DNA fragments may contain base changes resulting in mutant gene products. A comparison of specific activities of D-lactate dehydrogenase in the crude extracts of 50 recombinant clones indicated that one of the clones had drastically reduced enzyme activity. Nucleotide sequence analysis of the insert DNA showed an exchange of A to G at position 795 resulting in substitution of Glu264 to Gly in the D-lactate dehydrogenase. The purified mutant D-lactate dehydrogenase showed a shift of 2 units in its optimum pH toward the acidic range. The dependence of kcat/Km on the pH of the mutant enzyme showed that the pKa of the free enzyme was around 4, at least 2 pH units lower than that of the wild-type enzyme. Both the wild-type and the mutant enzyme at their respective optimum pH values showed similar kcat and Km values. The data suggest that the highly conserved Glu264 is not critical for enzyme catalysis, but it must be situated within hydrogen bonding distance to amino acid residue(s) involved in substrate binding as well as in catalysis.
Nestlé Research Centre, Vers-chez-les-Blanc, Lausanne, Switzerland.
S
S
Kochhar
N
N
Chuard
H
H
Hottinger
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000502:physiology*; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D045022:Lactate Dehydrogenases; D007778:Lactobacillus / Q000201:enzymology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011773:Pyruvates / Q000737:chemistry; D019289:Pyruvic Acid; D011994:Recombinant Proteins / Q000378:metabolism
D005971:Glutamates; D011773:Pyruvates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D019289:Pyruvic Acid; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase