O-phospho-L-serine and the thiocarboxylated sulfur carrier protein CysO-COSH are substrates for CysM, a cysteine synthase from Mycobacterium tuberculosis.
The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, was studied by transient-state kinetic techniques. The expression of which is upregulated under conditions of oxidative stress. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine sulfhydrylase family and employs a similar chemical mechanism involving a stable alpha-aminoacrylate intermediate. However, we show that specificity of CysM for its amino acid substrate is more than 500-fold greater for O-phospho-L-serine than for O-acetyl-L-serine, suggesting that O-phospho-L-serine is the likely substrate in vivo. We also investigated the kinetics of the carbon-sulfur bond-forming reaction between the CysM-bound alpha-aminoacrylate intermediate and the thiocarboxylated sulfur carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than 3 orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, while the reaction with bisulfide is consistent with a rapid equilibrium binding model. We interpret this finding to suggest that the CysM active site with the bound aminoacrylate intermediate is protected from solvent and that binding of CysO-COSH produces a conformational change allowing rapid sulfur transfer. This study represents the first detailed kinetic characterization of sulfide transfer from a sulfide carrier protein.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Seán E
SE
O'Leary
Christopher T
CT
Jurgenson
Steven E
SE
Ealick
Tadhg P
TP
Begley
D002352:Carrier Proteins / Q000378:metabolism; D003545:Cysteine / Q000096:biosynthesis; D003547:Cysteine Synthase / Q000378:metabolism*; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D018384:Oxidative Stress; D010768:Phosphoserine / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D002352:Carrier Proteins; D010768:Phosphoserine; D012694:Serine; D003547:Cysteine Synthase; C043943:O-acetylserine; D003545:Cysteine