Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308.
Cysteine-195 was previously identified as a probable active site residue in isocitrate lyase (ICL) from Escherichia coli ML308 [Nimmo, Douglas, Kleanthous, Campbell and MacKintosh (1989) Biochem. J. 261, 431-435]. This residue was replaced with serine and alanine residues by site-directed mutagenesis. The mutated genes expressed proteins with low but finite ICL activity, which co-migrated with wild-type ICL on both SDS/ and native PAGE. The mutant proteins were purified and characterized. Fluorimetry and c.d. in both the near- and the far-u.v. regions showed no differences between the mutants and wild-type ICL, indicating that the conformations of the three enzymes were very similar. ICL C195A (Cys-195-->Ala) and C195S (Cys-195-->Ser) showed 8.4-fold and 3.6-fold increases in the Km for isocitrate, while their kcat. values showed 30- and 100-fold decreases respectively. The effect of pH on the kinetic properties of the wild-type and mutant ICLs was investigated. The results showed that the response of the mutant enzymes to pH was simpler than that of the wild-type. For the mutants, ionisation of a group with a pKa of approx. 7.8 affected the Km for isocitrate and kcat.. For the wild-type enzyme, these parameters were affected by the ionization of two or more groups, one of which is presumed to by cysteine-195. The results are consistent with the view that the previously identified group with a pKa of 7.1 whose ionization affects the reaction of ICL by iodoacetate is cysteine-195 itself.
Robertson
A G
AG
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
Nimmo
H G
HG
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D001426:Bacterial Proteins; D007522:Isocitrate Lyase; D003545:Cysteine