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

Robertson, A.G.S. and Nimmo, H.G. (1995) Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308. Biochemical Journal, 305(1), pp. 239-244. (doi: 10.1042/bj3050239) (PMID:7826335) (PMCID:PMC1136455)

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Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Nimmo, Professor Hugh
Authors: Robertson, A.G.S., and Nimmo, H.G.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Biochemical Journal
Publisher:Portland Press
ISSN:0264-6021
ISSN (Online):1470-8728

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