Mechanistic Probes and Inhibitors of L - Pipecolate Oxidase

approved: T. Mark Zabriskie L-pipecolic acid (L-PA), a six carbon imino acid, is an intermediate of lysine degradation in various organisms including bacteria, yeast, fungi and mammals. In primates, the oxidation of L-PA by the enzyme L-pipecolate oxidase (L-PO) is the rate determining step of L-lysine degradation in the central nervous system. This thesis describes efforts to probe the L-PO mechanism by using different heteroatom substrate analogs as 'alternative substrates and inactivators of the enzyme. Thirteen heterocyclic analogs of L-PA containing nitrogen, oxygen and sulfur have been synthesized and tested as inhibitors and alternate substrates of L-PO. The oxygen analog, (R, S)-1,3-oxazine-4-carboxylic acid is neither an inhibitor nor a substrate of the enzyme, while nitrogen analogs such as (S)-3,4,5,6-tetrahydropyrimidine-4-carboxylic acid and (S)-hexahydropyrimidine-4-carboxylic acid proved to be weak substrates and inhibitors. Sulfur analogs have proved to be the best inhibitors and alternate substrates of L-PO. These include the mechanism-based inactivator and excellent substrate (S)-1,3thiazane-4-carboxylic acid. (S)-1,4-Thiazane-4-carboxylic acid is also an excellent substrate with a higher binding affinity for L-PO than the natural substrate. The turnover product of (S)-1,3-thiazane-4-carboxylic has been characterized by derivatization, HPLC analysis and mass spectrometry to be homocysteine. The turnover product of (S)-1,4-thiazane-2-carboxylic acid was also characterized and shown to be L-aamino-4-thioadipate-8-semialdehyde. Mechanistic Probes and Inhibitors of L-Pipecolate Oxidase