| Popis: |
Lactate dehydrogenase (d-lactate:oxidoreductase, EC 1.1.1.28) from the homolactate-fermenting fungus, Pythium debaryanum, has been purified to a homogeneous-preparation with a yield of 10 to 12%. The enzyme has a molecular weight of approximately 98,000 to 115,000 daltons. It is inhibited by very low concentrations of GTP. GDP, ITP, and ATP are considerably less effective than GTP. All nucleotides, except GTP and GDP, which inhibit the enzyme act competitively at the pyridine nucleotide active site or sites. Inhibition by GTP is allosteric. GTP increases the cooperative binding of NADH and d(-)-lactate but not the binding of NAD+ and pyruvate. The latter substrates bind noncooperatively. A physiological basis for the regulation of the enzyme is presented. GTP, presumably, modulates the enzyme to regulate the flow of pyruvate, via oxalacetate and P-enolpyruvate when there is a demand for glucogenetic metabolism. The kinetic mechanism of the enzyme has been deduced to be an obligatory ordered binary-binary process. Either NAD+ or NADH must first bind to the free enzyme before lactate or pyruvate can bind. From the GTP inhibition plots, two binding sites for NADH and four for lactate were estimated. Inhibition by GTP has been found to be widespread among d(-)-lactate dehydrogenases present in fungi belonging to the Oomycetes and Hypochytridiomycetes. |
