A general method for relieving substrate inhibition in lactate dehydrogenases.
The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure alpha-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate 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 KM for pyruvate. The kinetic and binding properties 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.
Hewitt
C O
CO
Molecular Recognition Centre and Department of Biochemistry,School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.
Eszes
C M
CM
Sessions
R B
RB
Moreton
K M
KM
Dafforn
T R
TR
Takei
J
J
Dempsey
C E
CE
Clarke
A R
AR
Holbrook
J J
JJ
D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000235:genetics; D019344:Lactic Acid / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000737:chemistry; D019289:Pyruvic Acid / Q000737:chemistry
D004791:Enzyme Inhibitors; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase