Why would enzyme rate of reaction decrease with increasing substrate concentrati - (Apr/14/2016 )

Hi all,

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 . But my enzyme's activity decreased at lower than this.

Any help? 

it may be feedback (product) inhibition.

if it is caused by the substrate then it may not be the substrate itself but rather the condition of the substrate (eg pH of the solution).

many moons ago, while working with g6pdh, we found inhibition by rudp. turned out that the rudp solution was altering the pH of the reaction. when we corrected for this, no inhibition was found.

mdfenko on Fri Apr 15 11:09:27 2016 said:

it may be feedback (product) inhibition.

 

if it is caused by the substrate then it may not be the substrate itself but rather the condition of the substrate (eg pH of the solution).

 

many moons ago, while working with g6pdh, we found inhibition by rudp. turned out that the rudp solution was altering the pH of the reaction. when we corrected for this, no inhibition was found.

i want to ask about the publication of that work mdfenko 
thnx 

ink12 on Thu Apr 14 16:58:02 2016 said:

Hi all,

 

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 . But my enzyme's activity decreased at lower than this.

 

Any help? 

 

How do you calculate the rate of the reaction ink12 
i am doing something similar with NADPH but with DFR enzyme and DHK substrate and i am new to this so i need to know how can i calculate the rate of the reaction to know which condition is better 

this is the reference to published article:

Grossman, A. and R.E. McGowan, "Regulation of Glucose-6-Phosphate Dehydrogenase in Blue-Green Algae", Plant Physiol., 55, 658-662 (1975)

keep in mind that the corrected results were published, not the erroneous ones. i don't remember if we made a statement about it (probably not).