Role of Histidine-152 in cofactor orientation in the PLP-dependent O-acetylserine sulfhydrylase reaction.

ABSTRACT

O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of l-cysteine, the replacement of the beta-acetoxy group of O-acetyl-l-serine (OAS) by a thiol. The 5'-phosphate of the PLP cofactor is very tightly bound to the enzyme; it accepts 8 hydrogen bonds from enzyme side chains and a pair of water molecules, and is in close proximity to a helix dipole. Histidine-152 (H152) is one of the residues that, via a water molecule, is responsible for positioning the 5'-phosphate. Mutation of H152 to alanine was predicted to increase the freedom of the 5'-phosphate, and as a result the cofactor, giving a decrease in the overall rate of the reaction. The H152A mutant enzyme was thus prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and (31)P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. UV-visible absorbance and visible CD spectra are consistent with a shift in the ketoeneamine to enolimine tautomeric equilibrium toward the neutral enolimine in the internal Schiff base of the free enzyme (ISB), the amino acid external Schiff base (ESB), and the alpha-aminoacrylate intermediate (AA). (31)P NMR spectra clearly indicate the presence of two conformers (presumably open and closed forms of the enzyme) that interconvert slowly on the NMR time scale in the ISB and ESB. Kinetic data suggest the decreased rate of the enzyme likely reflects a decrease in the amount of active enzyme as a result of an increased flexibility of the cofactor which results in substantial nonproductive binding of OAS in its external Schiff base, and a stabilization of the external Schiff bases of OAS and S-carboxynitrophenyl-l-cysteine. The nonproductive binding and stabilization of the external Schiff bases are thus linked to the shift in the tautomeric equilibrium and increase in the rate of interconversion of the open and closed forms of the enzyme. The location of the 5'-phosphate in the cofactor-binding site determines additional interactions between the cofactor and enzyme in the closed (ESB) form of the enzyme, consistent with an increased rate of interconversion of the open and closed forms of the enzyme upon increasing the rate of flexibility of the cofactor.