ENZYMES AND INHIBITORS OF L-PIPECOLIC ACID METABOLISM

The overall goals of the proposed research project are to address
fundamental questions related to the enzymology of L-pipecolic acid
metabolism in the mammalian brain and to develop potent, specific
inhibitors of its degradation. Such inhibitors represent a potential new
means for developing anticonvulsive agents and for studying GABA receptor
complexes. L-Pipecolic (L-PA) acid is a six-carbon cyclic amino acid, the
higher homologue of L-proline, and is a minor product of lysine metabolism
in various organisms and most mammalian tissues. The notable exception is
the brain, where lysine is primarily degraded to L-PA which has been shown
to possess both neuromodulating and anticonvulsive properties. A large
body of evidence supports an interaction of L-pipecolate and GABAergic
transmission, either at GABA receptors or through binding to its own
receptor and exerting an allosteric effect on the GABA complex. Hence, a
primary objective of this proposal is to define the features of the key
enzyme involved in L-pipecolic acid catabolism in the brain and use this
information to develop specific inactivators of it, thereby decreasing L-
PA degradation and elevating its concentration and neurological effects. In liver and kidney, the first step of L-PA degradation is known to be
species dependent, and is catalyzed by either a flavin-containing
peroxisomal oxidase or mitochondrial dehydrogenase. Rhesus monkey liver L-
pipecolate oxidase has been purified, but nothing is known about the
enzymology of L-PA metabolism in the brain or about the traits of the
mitochondrial enzyme from any source. Thus, the first specific goals of
this project will be to isolate and fully characterize mitochondrial L-
pipecolate oxidizing enzymes from rabbit kidney and brain, and the
peroxisomal enzyme from primate brain. This will generate data leading to
a better understanding of L-PA metabolism in general and presents a unique
opportunity to study different enzymes catalyzing the same reaction in
different mammalian species. Complete analysis of these enzymes will
include: determining physical and kinetic properties; detailed cofactor
studies; and stereochemical and mechanistic relatedness of the enzymes
isolated from the different species and organelles. The second specific aim of this project involves designing and
constructing modified substrates and potent inhibitors of L-PA oxidation.
The focus will be on mechanism-based inactivators designed to exploit
features characteristic of flavin-dependent amine oxidases. These
inactivators will serve as valuable probes of the mechanisms and active
site environments of the various L-pipecolate oxidizing enzymes.
Inhibitors designed to develop highly reactive radical or electrophilic
species could form covalent adducts with the target enzymes and permit
active site sequencing and comparisons. Potent inactivators may unveil a
new route to designing therapeutically useful anticonvulsants and the
pipecolate analogs developed in this work may serve as valuable
pharmacological ligands for studying and classifying GABA receptors.