Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases.
Nicotinamide adenine dinucleotide (NAD)-dependent d(minus)-and l(plus)-lactate dehydrogenases have been partially purified 89- and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for d(minus)-lactate dehydrogenase and 136,000 to 139,000 for l(plus)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by d(minus)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, l(plus)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. alpha-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevisiae.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000645:Ammonium Sulfate; D002458:Cell Fractionation; D002474:Cell-Free System; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D003300:Copper / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007773:Lactates / Q000096:biosynthesis*; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010376:Pediococcus / Q000201:enzymology* / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D013053:Spectrophotometry; D013237:Stereoisomerism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D007527:Isoenzymes; D007773:Lactates; D011773:Pyruvates; D013439:Sulfhydryl Reagents; D009243:NAD; D003300:Copper; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D008628:Mercury; D000645:Ammonium Sulfate