Isoforms of chicken triosephosphate isomerase are due to specific oxidation of cysteine126.
The electrophoretic isoforms of mammalian triosephosphate isomerase (TPI; EC 5.3.1.1) are due to deamidation at two Asn-Gly sites (Asn15 and Asn71). Deamidation of these two asparagines in the subunit-subunit interface of the isologous dimer appears to destabilize the dimer and initiate degradation of the protein. Chicken TPI contains a lysine substitution for Asn71, thus precluding this deamidation site. Nevertheless, the chicken enzyme exhibits three electrophoretic isoforms. This multiplicity is not the result of deamidation of the remaining Asn15 site, but due to a specific site which is highly susceptible to oxidation. The three isoforms of chicken TPI can be reduced to a single form in the presence of high concentrations of reducing agents (e.g., greater than 15 mM dithiothreitol or greater than 50 mM 2-mercaptoethanol) and are also generated when oxidizing agents, such as oxidized glutathione, are present. The oxidized isoforms exhibit lowered catalytic activity and are more susceptible to denaturation and proteolytic degradation than the native enzyme. Structural analysis of the isoforms by chemical cleavage at the cysteine peptide bonds with 2-nitro-5-thiocyanobenzoic acid and subsequently at the methionines with CNBr followed by peptide sequencing reveals that Cys126 is the site of the modification. Since the oxidized isoforms of chicken TPI accumulate in vivo during aging analogous to the deamidated isoforms from mammals, it appears that TPI is the first example of a protein which has evolved two specific types of weak links which may initiate turnover of the protein.
Tang
C Y
CY
Department of Biochemistry, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Yüksel
K U
KU
Jacobson
T M
TM
Gracy
R W
RW
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003545:Cysteine; D004795:Enzyme Stability; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D014305:Triose-Phosphate Isomerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D014305:Triose-Phosphate Isomerase; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide