Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803: Kinetic analysis of cleavage of the persulfide intermediate by chemical reductants

Elham Behshad, Sara E. Parkin, J. Martin Bollinger

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

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 ∼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 kcat suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that kcat is ∼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.

Original languageEnglish (US)
Pages (from-to)12220-12226
Number of pages7
JournalBiochemistry
Volume43
Issue number38
DOIs
StatePublished - Sep 28 2004

Fingerprint

Synechocystis
Reducing Agents
Dithiothreitol
Kinetics
Cysteine
Pyridoxal Phosphate
Catalysis
Mercaptoethanol
Sulfur
Sulfhydryl Compounds
cysteine desulfurase
persulfides
Rate constants
Galium
Enzymes
Chemical Models
Coenzymes
Accessories

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{bc1e1d3d0afb41ecaed544ef0e775a20,
title = "Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803: Kinetic analysis of cleavage of the persulfide intermediate by chemical reductants",
abstract = "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 ∼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 kcat suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that kcat is ∼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.",
author = "Elham Behshad and Parkin, {Sara E.} and Bollinger, {J. Martin}",
year = "2004",
month = "9",
day = "28",
doi = "10.1021/bi049143e",
language = "English (US)",
volume = "43",
pages = "12220--12226",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "38",

}

Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803 : Kinetic analysis of cleavage of the persulfide intermediate by chemical reductants. / Behshad, Elham; Parkin, Sara E.; Bollinger, J. Martin.

In: Biochemistry, Vol. 43, No. 38, 28.09.2004, p. 12220-12226.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mechanism of cysteine desulfurase Slr0387 from Synechocystis sp. PCC 6803

T2 - Kinetic analysis of cleavage of the persulfide intermediate by chemical reductants

AU - Behshad, Elham

AU - Parkin, Sara E.

AU - Bollinger, J. Martin

PY - 2004/9/28

Y1 - 2004/9/28

N2 - 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 ∼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 kcat suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that kcat is ∼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.

AB - 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 ∼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 kcat suggests that persulfide cleavage is, in fact, primarily rate-determining, and this conclusion is confirmed by the observation that kcat is ∼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.

UR - http://www.scopus.com/inward/record.url?scp=4644301523&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=4644301523&partnerID=8YFLogxK

U2 - 10.1021/bi049143e

DO - 10.1021/bi049143e

M3 - Article

C2 - 15379560

AN - SCOPUS:4644301523

VL - 43

SP - 12220

EP - 12226

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 38

ER -