Affinity labeling of a catalytic site, cysteine(247), in rat mercaptopyruvate sulfurtransferase by chloropyruvate as an analog of a substrate.
A bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (EC 2.8.1.2), is inactivated by 3-chloropyruvate, an analog of 3-mercaptopyruvate serving as a sulfur-donor and -acceptor substrate. To elucidate a reaction mechanism of the enzyme, the inactivation kinetic studies using 3-chloropyruvate were carried out. However, 3-chloropyruvate cannot be mixed with 3-mercaptopyruvate, 2-mercaptoethanol and thiosulfate because these substrates decompose 3-chloropyruvate. Thus, 3-mercaptopyruvate sulfurtransferase was incubated with 3-chloropyruvate, and then the remaining activity was measured separately in the assay system containing 3-mercaptopyruvate and 2-mercaptoethanol. The inactivation kinetics was analyzed by Kitz and Wilson method (J. Biol. Chem. 237 (1962) 3245-3248). The inactivation of mercaptopyruvate sulfurtransferase by 3-chloropyruvate proceeded in one-on-one manner and exhibited pseudo first-order kinetics with k(inact) = 0.068 +/- 0.003 min(-1) and K(I) = 4.0 +/- 0.2 mM (n = 3, mean +/- S.D.). Further, SH titration using DTNB revealed that MST was inactivated by 3-chloropyruvate in a 1:1 stoichiometry. Site-directed mutagenesis for binding sites of 3-mercaptopyruvate (Arg(187)-->Gly or Arg(196)-->Gly) (J. Biol. Chem. 271 (1996) 27395-27401) did not critically affect the inactivation. These findings suggest that 3-chloropyruvate behaves as an affinity label and directly tags the catalytic site, Cys(247). An ESI-LC/Q-TOF mass spectrometric study suggests that a pyruvate adduct is formed at Cys(247), which mimics a reaction intermediate.
Nagahara
Noriyuki
N
Department of Environmental Medicine, Nippon Medical School, Tokyo, Japan. noriyuki@nms.ac.jp
Sawada
Nori
N
Nakagawa
Toshio
T
D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D019289:Pyruvic Acid / Q000031:analogs & derivatives / Q000737:chemistry*; D051381:Rats; D013466:Sulfurtransferases / Q000737:chemistry*
D000345:Affinity Labels; D019289:Pyruvic Acid; D013466:Sulfurtransferases; C021550:3-mercaptopyruvate sulphurtransferase; D003545:Cysteine