Identification of the reactive cysteine in clostridial glutamate dehydrogenase by site-directed mutagenesis and proof that this residue is not strictly essential.
Cys320 of clostridial glutamate dehydrogenase, a residue close to the coenzyme binding site, has been replaced by serine. The mutant enzyme was successfully overproduced and purified by using the normal protocol for the wild-type enzyme and also behaved indistinguishably from wild-type enzyme on native and SDS-PAGE. The specific activity was significantly enhanced in assays at both pH 7 (+90%) and pH 8 (+38%). Detailed initial-rate kinetics revealed that at pH 7 this increase was mainly attributable to a higher maximum rate, since the Km values for both substrates were marginally increased. In the mutant enzyme the inactivating reaction with DTNB that characterizes the wild-type enzyme is completely eliminated. This proves that inactivation of the wild-type enzyme is due to modification of Cys320, that nevertheless Cys320 is not strictly essential for catalytic activity and that the remaining cysteine residue at position 144 is inaccessible to DTNB. Provision of an engineered subunit with a correct native structure but with its DTNB titre decreased from 1 to 0 mol/mol now offers a valuable tool for counting subunits in hybrid oligomers.
Wang
X G
XG
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Engel
P C
PC
D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D003545:Cysteine / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship
D012694:Serine; D004228:Dithionitrobenzoic Acid; D005969:Glutamate Dehydrogenase; D003545:Cysteine