Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803: kinetic analysis of cleavage of the persulfide intermediate by chemical reductants.
Cysteine desulfurases (CDs) are pyridoxal-5'-phosphate (PLP)-dependent enzymes that cleave sulfur from cysteine via an enzyme cysteinyl persulfide intermediate. In vitro studies of these enzymes have generally employed dithiothreitol as a cosubstrate to reductively cleave the persulfide intermediate, and it has been suggested that persulfide cleavage is the rate-limiting step for catalysis. In this study, the kinetics and mechanisms of cleavage of the persulfide intermediate in Slr0387 (CD-0387), a sequence group I (NifS/IscS-like) cysteine desulfurase from Synechocystis sp. PCC 6803, by physiological and nonphysiological reductants have been examined, and the extent to which this step is rate-limiting for catalysis has been determined. The observations that dithiols such as dithiothreitol (DTT) cleave the persulfide with approximately 100-fold greater efficiency than structurally similar monothiols such as 2-mercaptoethanol (2-ME), that cleavage by DTT exhibits saturation kinetics, and that the dependence of the observed first-order rate constant for persulfide cleavage by DTT on the concentration of the dithiol corresponds precisely with that for formation of a complex between DTT and the PLP cofactor of the resting enzyme suggest that persulfide cleavage by dithiols occurs by prior formation of a complex, in which addition of one thiol to the cofactor positions the second thiol for attack. This conclusion and the observation that a second molecule of L-cysteine can bind to the cofactor in the persulfide form of CD-0387 explain why several CDs are subject to potent inhibition by L-cysteine during turnover with DTT: binding of L-cysteine prevents formation of the PLP-DTT adduct and renders the dithiol no better than a monothiol, which must react with the persulfide in bimolecular fashion. Consistent with this rationale, catalysis by CD-0387 with 2-ME as cosubstrate, while less efficient, is not subject to potent inhibition by L-cysteine. The similarity of the maximum rate constant for persulfide cleavage by DTT to k(cat) suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that k(cat) is approximately 10-fold greater when tris-(2-carboxyethyl)phosphine (TCEP), the most efficient persulfide cleaver identified, is used as the reducing cosubstrate. The faster turnover with TCEP provides a chemical model for activation of CD-0387 and other CDs by the presence of accessory factors that serve as efficient acceptors of the persulfide sulfur.
Behshad
Elham
E
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Parkin
Sara E
SE
Bollinger
J Martin
JM
D013437:Carbon-Sulfur Lyases / Q000378:metabolism*; D002384:Catalysis; D000458:Cyanobacteria / Q000201:enzymology*; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D010720:Phosphines / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D019163:Reducing Agents / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013440:Sulfides / Q000737:chemistry* / Q000378:metabolism*; D013696:Temperature
D013437:Carbon-Sulfur Lyases; D003545:Cysteine; D004229:Dithiothreitol; D008623:Mercaptoethanol; D010720:Phosphines; D011732:Pyridoxal Phosphate; D019163:Reducing Agents; D013438:Sulfhydryl Compounds; D013440:Sulfides; C051552:persulfides; C080938:tris(2-carboxyethyl)phosphine; C080546:cysteine desulfurase