Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)

Fan Fan, Andreas Luxenburger, Gavin F. Painter, John S. Blanchard

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido- 2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s -1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of ∼306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of ∼9.4 and ∼5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.

Original languageEnglish (US)
Pages (from-to)11421-11429
Number of pages9
JournalBiochemistry
Volume46
Issue number40
DOIs
StatePublished - Oct 9 2007

Fingerprint

Mycobacterium smegmatis
Ligases
Cysteine
Melanocyte-Stimulating Hormones
Adenosine Triphosphate
Kinetics
Actinobacteria
Biosynthesis
Biosynthetic Pathways
Cell growth
Enzymes
Adenosine Monophosphate
Mycobacterium tuberculosis
Kinetic parameters
Adenosine
Condensation
Oxidative Stress
Anti-Bacterial Agents
Inhibition (Psychology)
Growth

ASJC Scopus subject areas

  • Biochemistry

Cite this

Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC). / Fan, Fan; Luxenburger, Andreas; Painter, Gavin F.; Blanchard, John S.

In: Biochemistry, Vol. 46, No. 40, 09.10.2007, p. 11421-11429.

Research output: Contribution to journalArticle

Fan, Fan ; Luxenburger, Andreas ; Painter, Gavin F. ; Blanchard, John S. / Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC). In: Biochemistry. 2007 ; Vol. 46, No. 40. pp. 11421-11429.
@article{1fca93498f8d40e890da334e21259a0c,
title = "Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)",
abstract = "Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido- 2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s -1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of ∼306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of ∼9.4 and ∼5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.",
author = "Fan Fan and Andreas Luxenburger and Painter, {Gavin F.} and Blanchard, {John S.}",
year = "2007",
month = "10",
day = "9",
doi = "10.1021/bi7011492",
language = "English (US)",
volume = "46",
pages = "11421--11429",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "40",

}

TY - JOUR

T1 - Steady-state and pre-steady-state kinetic analysis of Mycobacterium smegmatis cysteine ligase (MshC)

AU - Fan, Fan

AU - Luxenburger, Andreas

AU - Painter, Gavin F.

AU - Blanchard, John S.

PY - 2007/10/9

Y1 - 2007/10/9

N2 - Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido- 2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s -1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of ∼306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of ∼9.4 and ∼5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.

AB - Mycobacterium tuberculosis and many other members of the Actinomycetes family produce mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido- 2-deoxy-α-D-glucopyranoside (MSH or AcCys-GlcN-Ins), to act against oxidative and antibiotic stress. The biosynthesis of MSH is essential for cell growth and has been proposed to proceed via a biosynthetic pathway involving four key enzymes, MshA-MshD. The MSH biosynthetic enzymes present potential targets for inhibitor design. With this as a long-term goal, we have carried out a kinetic and mechanistic characterization, using steady-state and pre-steady-state approaches, of the recombinant Mycobacterium smegmatis MshC. MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins. Initial velocity and inhibition studies show that the steady-state kinetic mechanism of MshC is a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding followed by cysteine binding, release of PPi, binding of GlcN-Ins, followed by the release of Cys-GlcN-Ins and AMP. The steady-state kinetic parameters were determined to be kcat equal to 3.15 s -1, and Km values of 1.8, 0.1, and 0.16 mM for ATP, cysteine, and GlcN-Ins, respectively. A stable bisubstrate analogue, 5′-O-[N-(L-cysteinyl)sulfamonyl]adenosine, exhibits competitive inhibition versus ATP and noncompetitive inhibition versus cysteine, with an inhibition constant of ∼306 nM versus ATP. Single-turnover reactions of the first and second half reactions were determined using rapid-quench techniques, giving rates of ∼9.4 and ∼5.2 s-1, respectively, consistent with the cysteinyl adenylate being a kinetically competent intermediate in the reaction by MshC.

UR - http://www.scopus.com/inward/record.url?scp=35048813938&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=35048813938&partnerID=8YFLogxK

U2 - 10.1021/bi7011492

DO - 10.1021/bi7011492

M3 - Article

VL - 46

SP - 11421

EP - 11429

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 40

ER -