Enzymes containing the cofactor, pyridoxal phosphate, are ubiquitous in biology, performing essential functions in the metabolism of amino acids and amines. These enzymes catalyze a wide variety of reactions, including transaminations, racemizations, alpha- and beta-decarboxylations, retro-Aldol cleavages, beta- and gamma-eliminations and substitutions. The goal of our research is to understand, at the molecular level, the mechanisms of three of these enzymes, tryptophan indole-lyase, tyrosine phenol-lyase, and kynureninase. These enzymes catalyze unusual elimination reactions with carbon leaving groups, and thus require chemical steps involving the leaving groups in order for the reactions to proceed. We will synthesize and evaluate novel mechanism-based inhibitors and suicide substrates for these enzymes. In addition, we will prepare and examine a series of aza-analogues of substrates for tryptophan indole-lyase and tyrosine phenol-lyase. We will demonstrate the reversibility of the reaction of kynureninase using aryl esters to catalyze Claisen-type condensations with L-alanine. We will perform both steady-state and stopped-flow kinetic studies with these enzymes. For kynureninase, we will evaluate pH dependencies and isotope effects on the steady-state kinetics to evaluate the role of general acid/base catalysis. We will also examine in detail the effects of monovalent cations on the steady-state and pre-steady-state kinetic parameters for tryptophan indole-lyase and tyrosine phenol-lyase. These data will provide important information about the details of the enzymatic reaction mechanisms. We will clone and sequence the gene coding for tyrosine phenol-lyase from Citrobacter freundii. Then we will be able to determine the homology of tyrosine phenol-lyase with tryptophan indole-lyase. This will allow us to determine the evolutionary relationships between the enzymes with respect to structure, mechanism and regulation. Also, we will prepare mutants of tryptophan indole-lyase by sitedirected mutagenesis. These studies will allow us to evaluate the structural and catalytic roles of specific amino acid residues.
| Phillips, Robert S (2015) Chemistry and diversity of pyridoxal-5'-phosphate dependent enzymes. Biochim Biophys Acta 1854:1167-74 |
| Phillips, Robert S; Demidkina, Tatyana V; Faleev, Nicolai G (2014) The role of substrate strain in the mechanism of the carbon-carbon lyases. Bioorg Chem 57:198-205 |
| Phillips, Robert S (2014) Structure and mechanism of kynureninase. Arch Biochem Biophys 544:69-74 |
| Phillips, Robert S (2011) Structure, mechanism, and substrate specificity of kynureninase. Biochim Biophys Acta 1814:1481-8 |
| Lima, Santiago; Khristoforov, Roman; Momany, Cory et al. (2007) Crystal structure of Homo sapiens kynureninase. Biochemistry 46:2735-44 |
