DIFFERENTIAL INACTIVATION OF RABBIT AND YEAST TRIOSEPHOSPHATE ISOMERASE - EFFECT OF OXIDATIONS PRODUCED BY CHLORAMINE-T

Triosephosphate isomerase from rabbit has 5 Cys and 2 Met, while trios ephosphate isomerase from yeast has 2 Cys (present in the rabbit enzym e in equivalent positions) and no Met. Since chloramine-T oxidizes Cys and Met, we determined the effect it has on the activity and structur e of both enzymes. The activity of triosephosphate isomerase from rabb it was more sensitive to chloramine-T than that of the yeast enzyme (u nder conditions where the rabbit isomerase was completely inactive, th e yeast enzyme exhibited approximately 50% activity). An initial effec t of chloramine-T on triosephosphate isomerase was the oxidation of Cy s and the formation of catalytically active acidic isoforms. For the y east isomerase, the two processes were slower. Our data suggest that o xidation of Cys 126, which is conserved in all of the studied species, does not abolish catalysis. Chloramine-T also oxidized the two Met of the rabbit enzyme. At ratios of 50 chloramine-T/monomer, circular dic hroism studies showed that the rabbit enzyme, but not that from yeast, underwent extensive alterations of tertiary and secondary structures. This was accompanied by formation of stable dimers, whose cross-linki ng was not through disulfide bonds. Studies of dimer formation at vari ous enzyme concentrations showed that cross-linking was between monome rs of the same dimer. Under conditions that led to cross-linking, rabb it triosephosphate isomerase took up 2.7 mol of H-3 from (NaBH4)-H-3/m ol dimer, and the yeast enzyme incorporated only 0.4 mol of H-3. Thus cross-linking was most likely via a Schiff base. The results revealed the points whose modification caused inactivation of the rabbit enzyme . (C) 1994 Academic Press, Inc.