Steve Ealick's Research Group

Abstract:

O'Leary S, Jurgenson CT, Ealick SE, and Begley TP. O-Phospho-L-serine and the Thiocarboxylated Sulfur Carrier Protein CysO-COSH are Substrates for CysM, a Cysteine Synthase from Mycobacterium tuberculosis. Biochemistry 47:11606-11615 (2008).

The kinetic pathway of CysM, a cysteine synthase from Mycobacterium tuberculosis, the expression of which is upregulated under conditions of oxidative stress, was studied by transient-state kinetic techniques. This enzyme exhibits extensive homology with the B-isozymes of the well-studied O-acetylserine
sulfhydrylases and employs a similar chemical mechanism involving a stable α- 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 α-aminoacrylate intermediate and the thiocarboxylated sulfur-carrier protein, CysO-COSH. The specificity of CysM for this physiological sulfide equivalent is more than three orders of magnitude greater than that for bisulfide. Moreover, the kinetics of this latter reaction are limited by association of the proteins, whilst 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.