The catalytic role of aspartate in the active site of glutamate dehydrogenase.
A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by site-directed mutagenesis. The mutant enzyme is efficiently overexpressed in Escherichia coli as a soluble protein and can be successfully purified by the dye-ligand chromatographic procedure normally employed for the wild-type enzyme. By several criteria, including circular dichroism spectrum, sulphydryl reactivity with Ellman's reagent, crystallization and mobility in non-denaturing electrophoresis, the enzyme appears to be correctly folded. NAD+ protects the D165S mutant against modification by Ellman's reagent, suggesting unimpaired binding of coenzyme. In standard assays the specific activity is decreased 10(3)-fold in the reductive amination reaction and 10(5)-fold for oxidative deamination. Kinetic studies show that apparent Km values for NADH and 2-oxoglutarate are almost unchanged. The large reduction in the reaction rate coincides with a weakening of the affinity for ammonium ion (Km > 300 mM, compared with 60 mM for the wild-type). The data are entirely consistent with the direct involvement of D165 in catalysis rather than in the binding of coenzyme or 2-oxoglutarate.
Dean
J L
JL
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Wang
X G
XG
Teller
J K
JK
Waugh
M L
ML
Britton
K L
KL
Baker
P J
PJ
Stillman
T J
TJ
Martin
S R
SR
Rice
D W
DW
Engel
P C
PC
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D004269:DNA, Bacterial; D001224:Aspartic Acid; D005969:Glutamate Dehydrogenase