O-phospho-L-serine and the thiocarboxylated sulfur carrier protein CysO-COSH are substrates for CysM, a cysteine synthase from Mycobacterium tuberculosis

Abstract

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.

Scheme 1

Biosynthetic pathways for L-cysteine

Scheme 2

Proposed chemical mechanism for CysM

Scheme 3

Minimal kinetic pathway and determined rate constants for CysM

Figure 1

Ultraviolet-visible spectroscopy of formation of the CysM-bound α-aminoacrylate intermediate. (A) Mixing of CysM (14 μM) with O-acetyl-L-serine (5 mM) produces a decrease in the absorbance due to enzyme-bound pyridoxal-5′-phosphate (1) at 412 nm and an increase in the absorbance at 458 nm due to the formation of an α-aminoacrylate intermediate (5). The traces were recorded at intervals of approximately 34 s after mixing, in 50 mM Tris-HCl, pH 8.0 and at room temperature. (B) Absorbance changes due to mixing of O-phospho-L-serine (10 μM) with CysM (14 μM) in 50 mM Tris-HCl at pH 8.0 and at room temperature. Individual traces were recorded at intervals of approximately 30 s. The changes reflect conversion of the enzyme-bound PLP imine (1, λ_max = ∼412 nm) to a stable α-aminoacrylate intermediate (5, λ_max = ∼462 nm).

Figure 2

Kinetics of formation of α-aminoacrylate intermediate from O-phospho-L-serine. (A) Changes in absorbance at 465 nm following rapid mixing of O-phospho-L-serine (OPS; 0.5, 1, 2, 5 and 10 mM in order of increasing rate) with CysM (4.5 μM) in 50 mM Tris-HCl at pH 8 and at ∼22 °C. The resulting data were fit to single exponential functions to give the first order rates at the various concentrations of O-phospho-L-serine. (B) Plot of the rates of acrylate formation as a function of O-phospho-L-serine concentration, fit to a hyperbolic function describing a rapid equilibrium binding model. From this fit, the K_d for OPS was found to be 6 ± 1 mM and the second order rate constant for formation of the aminoacrylate intermediate was 2.8 ± 0.7 mM^-1 s^-1.

Figure 3

Kinetics of formation and decay of the α-aminoacrylate intermediate in the presence of cysteine. (A) CysM (31 μM) was mixed with cysteine (1, 4 and 8 mM in order of increasing rate) in 50 mM Tris-HCl, pH 8.0 and at ∼22 °C and the change in absorbance at 465 nm was monitored. The data are shown fit to functions of the form y = Ae^-kt + Be^-kt + C. (B) Dependence of the rate of formation of the aminoacrylate intermediate on the concentration of cysteine. The slope of the line fitting the data is a measure of the specificity of the enzyme for this amino acid substrate and was found to be 0.0044 ± 0.0004 mM^-1 s^-1. The rate of decay of the aminoacrylate intermediate was independent of the concentration of L-cysteine within experimental error, being of the order of 0.001 s^-1.

Figure 4

Kinetics of quenching of the α-aminoacrylate intermediate by bisulfide. The CysM/O-phospho-L-serine α-aminoacrylate intermediate was pre-formed by mixing CysM with a stoichiometric quantity of O-phospho-L-serine in 50 mM Tris-HCl, pH 8.0 at room temperature (∼22 °C). The solution containing the intermediate was then mixed with a solution of sodium sulfide in the same buffer, resulting in reaction of the α-aminoacrylate intermediate. The final solution contained the reducing agent tris(2-carboxyethyl)phosphane (TCEP) at a concentration of 2 mM. (A) Representative trace with exponential fit showing decay of absorbance at 465 nm after rapid mixing of the pre-formed CysM/O-phospho-L-serine α-aminoacrylate intermediate (11 μM) with sodium sulfide (0.5 mM). (B) Plot of the first order rates of quenching extracted from the exponential fits at various bisulfide concentrations (0.1 – 15 mM), fit to a hyperbolic function, which describes a rapid equilibrium binding model. The K_d for bisulfide was found to be 7 ± 2 mM and the first order rate constant for the carbon-sulfur bond-forming addition reaction was 0.48 ± 0.07 s^-1.

Figure 5

Kinetics of quenching of the α-aminoacrylate intermediate by CysO-COSH. The CysM/O-phospho-L-serine α-aminoacrylate intermediate was pre-formed by mixing CysM (14 μM) with O-phospho-L-serine (11 μM) in 50 mM Tris-HCl, pH 8.0 at room temperature (∼22 °C). This solution was then rapidly mixed with a solution containing CysO-COSH in the same buffer. (A) Representative trace with exponential fit showing decay of absorbance at 465 nm after rapid mixing of the CysM/O-phospho-L-serine α-aminoacrylate intermediate with CysO-COSH (0.05 mM) in 50 mM Tris-HCl at pH 8 and at room temperature. (B) Plot of the first-order rates of decay at the various CysO-COSH concentrations with a linear fit. The slope of this line is a measure of the specificity of the enzyme for this nucleophilic substrate and was found to be 88 ± 6 mM^-1 s^-1.

Figure 6

Stereoview of the active site model of the O-phospho-L-serine-PLP imine at the active site of CysM. The O-phospho-L-serine-PLP imine is indicated with cyan carbon atoms.