SITE-DIRECTED MUTAGENESIS OF CYSTEINE-195 IN ISOCITRATE LYASE FROM ESCHERICHIA-COLI ML308

Cysteine-195 was previously identified as a probable active site resid ue in isocitrate lyase (ICL) from Escherichia coli ML308 [Nimmo, Dougl as, Kleanthous, Campbell and MacKintosh (1989) Biochem. J. 261, 431-43 5]. This residue was replaced with serine and alanine residues by site -directed mutagenesis. The mutated genes expressed proteins with low b ut finite ICL activity, which co-migrated with wild-type ICL on both S DS/ and native PAGE. The mutant proteins were purified and characteriz ed. Fluorimetry and c.d. in both the near- and the far-u.v. regions sh owed no differences between the mutants and wild-type ICL, indicating that the conformations of the three enzymes were very similar. ICL C19 5A (Cys-195-->Ala) and C195S (Cys-195-->Ser) showed 8.4-fold and 3.6-f old increases in the K-m for isocitrate, while their k(cat.) values sh owed 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 s impler than that of the wild-type. For the mutants, ionisation of a gr oup 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 cyste ine-195. The results are consistent with the view that the previously identified group with a pK(a) of 7.1 whose ionization affects the reac tion of ICL by iodoacetate is cysteine-195 itself.