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Why would enzyme rate of reaction decrease with increasing substrate concentrati

enzyme kinetics rate of reaction enzyme substrate

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#1 ink12

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Posted 14 April 2016 - 08:58 AM

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

 

Any help? 

 



#2 mdfenko

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Posted 15 April 2016 - 03:09 AM

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.


Edited by mdfenko, 15 April 2016 - 03:13 AM.

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