Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308

Abstract

Cysteine-195 was previously identified as a probable active site residue in isocitrate lyase (ICL) from Escherichia coli ML308. 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 K(m) for isocitrate, while their k(cat.) 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 pK(a) of approx. 7.8 affected the K(m) for isocitrate and k(cat.) For the wild-type enzyme, these parameters were affected by the ionization of two or more groups, one of which is presumed to be cysteine-195. The results are consistent with the view that the previously identified group with a PK(a) of 7.1 whose ionization affects the reaction of ICL by iodoacetate is cysteine-195 itself.