A general method for relieving substrate inhibition in lactate dehydrogenases

The mutation S163L in human heart lactate dehydrogenase removes substrate i nhibition while only modestly reducing the turnover rate for pyruvate, Sinc e this is the third enzyme to show this behaviour, we suggest that the S163 L mutation is a general method for the removal of substrate inhibition in L -LDH enzymes. Engineering such enzymatic properties has clear industrial ap plications in the use of these enzymes to produce enantiomerically pure alp ha-hydroxy acids. The mutation leads to two principal effects, (1) Substrat e inhibition is caused by the formation of a covalent adduct between pyruva te and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD(+) binary complex is not perturbed. (2) The mutation also leads to a large increase in the K-M for pyruvate, The kinetic and binding proper ties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme-NADH complex.