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 triosephosphate isomerase from yeast has 2 Cys (present in the rabbit enzyme in equivalent positions) and no Met. Since chloramine-T oxidizes Cys and Met, we determined the effect it has on the activity and structure of both enzymes. The activity of triosephosphate isomerase from rabbit was more sensitive to chloramine-T than that of the yeast enzyme (under conditions where the rabbit isomerase was completely inactive, the yeast enzyme exhibited approximately 50% activity). An initial effect of chloramine-T on triosephosphate isomerase was the oxidation of Cys and the formation of catalytically active acidic isoforms. For the yeast isomerase, the two processes were slower. Our data suggest that oxidation 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 dichroism 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-linking was not through disulfide bonds. Studies of dimer formation at various enzyme concentrations showed that cross-linking was between monomers of the same dimer. Under conditions that led to cross-linking, rabbit triosephosphate isomerase took up 2.7 mol of 3H from NaB3H4/mol dimer, and the yeast enzyme incorporated only 0.4 mol of 3H. Thus cross-linking was most likely via a Schiff base. The results revealed the points whose modification caused inactivation of the rabbit enzyme.
Zubillaga
R A
RA
Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, México, D.F.
Pérez-Montfort
R
R
Gómez-Puyou
A
A
D000818:Animals; D002700:Chloramines / Q000494:pharmacology*; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis; D004228:Dithionitrobenzoic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007700:Kinetics; D008715:Methionine / Q000032:analysis; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation / Q000187:drug effects*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D014105:Tosyl Compounds / Q000494:pharmacology*; D014305:Triose-Phosphate Isomerase / Q000037:antagonists & inhibitors* / Q000737:chemistry
D002700:Chloramines; D007202:Indicators and Reagents; D010446:Peptide Fragments; D014105:Tosyl Compounds; C016300:chloramine-T; D004228:Dithionitrobenzoic Acid; D008715:Methionine; D014305:Triose-Phosphate Isomerase; D003545:Cysteine; D003488:Cyanogen Bromide