Elucidation of the Stereochemical Mechanism of Cystathionine γ-Lyase Reveals How Substrate Specificity Constrains Catalysis

Abstract

Pyridoxal phosphate (PLP)-dependent enzymes play essential roles in metabolism and have found applications for organic synthesis and as enzyme therapeutics. The vinylglycine ketimine (VGK) subfamily hosts a growing set of enzymes that play diverse roles in primary and secondary metabolism. However, the molecular determinants of substrate specificity and the complex acid−base chemistry that enables VGK catalysis remain enigmatic. We use a recently discovered amino acid γ-lyase as a model system to probe catalysis in this enzyme family. We discovered that two stereochemically distinct proton transfer pathways occur. Combined kinetic and spectroscopic analysis revealed that progression through the catalytic cycle is correlated with the presence of an Hbond donor after Cγ of an amino acid substrate, suggesting that substrate binding is kinetically coupled to a conformational change. High-resolution X-ray crystallography shows that cystathionine-γ-lyases generate an s-trans intermediate and that this geometry is likely conserved throughout the VGK family. An H-bond acceptor in the active site templates substrate binding but does so by preorganizing substrates away from catalytically productive orientations. Mutagenesis eliminates this preorganization, such that there is a relaxation of the substrate specificity but an increase in k cat for diverse substrates. We exploit this information to perform a preparative-scale α,β,β-trideuteration of polar amino acids. Together, these data untangle a complex mode of substrate specificity and provide a foundation for the future study and applications of VGK enzymes.