Effect of an arginine-to-isoleucine active site mutation on Escherichia coli malate dehydrogenase enzymatic activity

Citric acid cycle enzymes function in an environment with numerous substrate analogues and therefore contain active site residue organizations that confer high substrate specificity. Extensive research into the catalytic mechanism of Escherichia coli malate dehydrogenase (eMDH) has identified arginine81 as being crucial to catalysis. In this investigation, an engineered eMDH having an Ile81 rather than an Arg81 (R81I) was isolated using a hexahistadine (His6) tag. Enzymatic activity of the R81I mutant with respect to malate, lactate, and pyruvate was explored. The R81I mutant did show significant activity toward malate, but did not show significant activity toward lactate or pyruvate. Investigations into an R81F mutant eMDH and an R81W/M85E dimutant eMDH may provide more insight into the eMDH catalytic mechanism