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 [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.

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

“…This cysteine is strictly conserved in all known ICL, consistent with its crucial role in catalysis (29,30,50,51). This cysteine was proposed to be required for deprotonation of a carboxylate of succinate during the catalytic reaction (52).…”

“…1A). Cys 301 is only conserved in bacterial-type ICLs, whereas Cys 178 is conserved in all organisms, consistent with its role in catalysis (29,30) Because two cysteine residues of ICL have been previ-ously suggested to be sensitive to redox modifications (10,11,17), based on sequence conservation, Cys 178 and Cys 301 appear as the most likely candidates. Three-dimensional modeling of CrICL suggests that these two residues are the two closest cysteines in the enzyme but are too distant to allow formation of a disulfide bridge (Fig.…”

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

“…If the far UV and near UV CD spectra of the mutants are superimposable on those of the wild type protein, it can be concluded that the mutations have not resulted in any significant distortion of structure. This was found to be the case in two mutant forms of isocitrate lyase from Escherichia coli in which Cys 195 had been replaced by alanine or serine, leading to loss of activity [24] (see Fig. 2).…”

“…The far UV (a) and near UV (b) spectra for wild type enzyme and a mutant in which Cys 195 has been replaced by Ala are shown as solid and dashed lines respectively. The spectra of the mutant in which Cys 195 has been replaced by Ser are superimposable on those shown [24]. measurements in order to explore the relationships between quaternary and secondary or tertiary structures [26].…”

ALG2, the Hansenula polymorpha isocitrate lyase gene

“…In Alg2p, highly conserved regions are revealed, such as the hexapeptides KKCGHM (ICL signature; Figure 2) and SPSFNW (active site signature; Figure 2). The former contains a Cys (Cys 213 ), homologous to S. cerevisiae Cys 195 and to E. coli Cys 217 , the significance of which has been discussed (Ko and McFadden, 1990; Rehman and McFadden, 1997; Robertson and Nimmo, 1995); the latter contains the sequence SPS, which is likely to take part in the formation of the enzyme–substrate bond (Ko et al , 1992). Towards the amino‐terminus (residues 32–40), this protein contains a short stretch (RLEGHQESL) rather similar to the consensus sequence for PTS2, one of the microbody (peroxisomal) targeting signals (see Subramani, 1998).…”