I have a purified lactate dehydrogenase that proceed with this reaction: pyruvate + NADPH ⇌ d-lactate + NADP. It has a preference for the forward reaction and less so for the reverse reaction. Additionally, it can also catalyse this reaction: glyoxylate + NADPH ⇌ glycolate + NADP, but to a much lesser extent. I did some kinetic studies on these 3 reactions (pyruvate to lactate, lactate to pyruvate and glyoxylate to glycolate) using a phosphate buffer at pH 7.5.
The rate of reaction catalysing pyruvate → lactate increases with substrate concentration and then plateaus. However, the rate of reaction for lactate → pyruvate, and glyoxylate → glycolate increase until a certain point and then suddenly decreased when the concentration of substrate went too high. Is there any explanation for this? Could this be substrate inhibition? What can I do with my data for the 2 reactions to calculate reliable Km?
Also, if this is of importance, the lactate dehydrogenase originally used NADH/NAD as the cofactor but this was mutated to use NADPH/NADP instead. The reactions went well with the original enzyme and had none of this trouble except with glyoxylate when it did show decreasing activity at extremely high [glyoxylate]. But my enzyme's activity decreased at lower [glyoxylate] than this.