Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies.
A highly conserved lysine at position 128 of Escherichia coli glutamate dehydrogenase (GDH) has been altered by site-directed mutagenesis of the gdhA gene. Chemical modification studies have previously shown the importance of this residue for catalytic activity. We report the properties of mutants in which lysine-128 has been changed to histidine (K128H) or arginine (K128R). Both mutants have substantially reduced catalytic centre activities and raised pH optima for activity. K128H also has increased relative activity with amino acid substrates other than glutamate, especially L-norvaline. These differences, together with alterations in Km values, Kd values for NADPH and Ki values for D-glutamate, imply that lysine-128 is intimately involved in either direct or indirect interactions with all the substrates and also in catalysis. These multiple interactions of lysine-128 explain the diverse effects of chemical modifications of the corresponding lysine in homologous GHDs. In contrast, lysine-27, another highly reactive residue in bovine GDH, is not conserved in all of the sequenced NADP-specific GDHs and is therefore not likely to be involved in catalysis.
McPherson
M J
MJ
Biotechnology Unit, University of Leeds, UK.
Baron
A J
AJ
Jones
K M
KM
Price
G J
GJ
Wootton
J C
JC
D000595:Amino Acid Sequence; D002384:Catalysis; D004269:DNA, Bacterial / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000302:isolation & purification; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides; D010957:Plasmids; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009841:Oligonucleotides; D005969:Glutamate Dehydrogenase; D008239:Lysine