Species‐Specific Inhibition of Homologous Enzymes by Modification of Nonconserved Amino Acids Residues

Abstract

The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with triosephosphate isomerase. S-phenylp-toluenethiosulfonate (MePhS0,-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit triosephosphate isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit triosephosphate isomerase. The effect of MePhS0,-SPh on triosephosphate isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhS0,-SPh did not affect the activity of triosephosphate isomerase from S. cerevisiae and E. coli but it inhibited triosephosphate isomerase from chicken and S. pornhe. From an analysis of the Cys content of the various triosephosphate isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSOz-SPh. Methyl metanethiosulfonate (MeS0,-SMe), which produces methyl disulfides, had no effect on triosephosphate isomerases that lack Cys217 (S. cerevisiae and E. coli). In triosephosphate isomerases that have Cys217, MeS0,-SMe inhibited by 40-50% the activity of that from S. pornbe, 20-25% that from rabbit but had no effect on the chicken enzyme. In the three latter triosephosphate isomerases, MeS0,-SMe protected against the strong inhibiting action of MePhS0,-SPh. The latter observations suggest that MeS0,-SMe and MePhS0,-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit triosephosphate isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K,, for glyceraldehyde 3-phosphate and a 65% decrease in V,,,,,. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cysl4 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.

Disulfide Bridges in the Mesophilic Triosephosphate Isomerase from Giardia lamblia Are Related to Oligomerization and Activity

“…54 For the determination of kinetic constants, the concentration of GAP ranged from 0.1 mM to 3 mM. Kinetic parameters were calculated from initial velocities at each concentration of substrate.…”

Cloning, Expression, Purification and Characterization Of Triosephosphate Isomerase from Trypanosoma Cruzi

“…The sensitivity to MeS0,-SMe of various triosephosphate isomerases can be used to determine if a particular triosephosphate isomerase possesses a cysteine at position 14 [ I l , 221. For instance, triosephosphate isomerase from E. coli that lacks Cys14 is insensitive to MeS0,-SMe [22], whereas the activity of triosephosphate isomerases that have this residue are completely inhibited. Triosephosphate isomerase from 7: cruzi has this residue ( Fig.…”

Structural and biochemical characterization of a recombinant triosephosphate isomerase from Rhipicephalus (Boophilus) microplus

“…The kinetic constants Km and Vmax were measured, with glyceraldehyde 3-phosphate as substrate, these values were 0.48 mM and 6031 mmol/min  mg protein, respectively. The kinetic values are very similar to other TIMs of many organisms such as: rabbit, Saccharomyces cerevisiae, E. coli, chicken and Schizosaccharomyces pombe (Garza-Ramos et al, 1996).…”

“…Considering TIM as a target for drug development against various endoparasites associated with human diseases, such as Plasmodium, Trypanosoma cruzi, Trypanosoma brucei and Giardia lamblia (Garza-Ramos et al, 1996;Maldonado et al, 1998;Zomosa-Signoret et al, 2003), the rationale for drug discovery is based mainly on the identification and structural characterization of non-conserved amino acids that are essential in catalysis or stability of the enzymes from the parasite (Gomez-Puyou et al, 1995).…”