A general method for relieving substrate inhibition in lactate dehydrogenases

The mutation S163L in human heart lactate dehydrogenase removessubstrate inhibition while only modestly reducing the turnoverrate for pyruvate. Since this is the third enzyme to show thisbehaviour, we suggest that the S163L mutation is a general methodfor the removal of substrate inhibition in L-LDH enzymes. Engineeringsuch enzymatic properties has clear industrial applicationsin the use of these enzymes to produce enantiomerically pure-hydroxy acids. The mutation leads to two principal effects.(1) Substrate inhibition is caused by the formation of a covalentadduct between pyruvate and the oxidized form of the cofactor.The inability of S163L mutants to catalyse the formation ofthis inhibitory adduct is demonstrated. However, NMR experimentsshow that the orientation of the nicotinamide ring in the mutantNAD⁺ binary complex is not perturbed. (2) The mutation alsoleads to a large increase in the K_M for pyruvate. The kineticand binding properties of S163L LDH mutants are accounted forby a mechanism which invokes a non-productive, bound form ofthe cofactor. Molecular modelling suggests a structure for thisnon-productive enzyme–NADH complex.