THE CATALYTIC ROLE OF ASPARTATE IN THE ACTIVE-SITE OF GLUTAMATE-DEHYDROGENASE

被引:28
作者
DEAN, JLE
WANG, XG
TELLER, JK
WAUGH, ML
BRITTON, KL
BAKER, PJ
STILLMAN, TJ
MARTIN, SR
RICE, DW
ENGEL, PC
机构
[1] UNIV SHEFFIELD,KREBS INST BIOMOLEC RES,DEPT MOLEC BIOL & BIOTECHNOL,SHEFFIELD S10 2TN,S YORKSHIRE,ENGLAND
[2] NATL INST MED RES,DIV PHYS BIOCHEM,LONDON NW7 1AA,ENGLAND
基金
英国惠康基金;
关键词
D O I
10.1042/bj3010013
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 K-m 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 (K-m > 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.
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收藏
页码:13 / 16
页数:4
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