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Research
Research in our laboratory involves synthetic and
mechanistic organic chemistry employed in combination with
mechanistic biochemistry. Three different types of enzymes
have been selected for study: folypoly-g-glutamate
synthetase/g-glutamyl hydrolase, glycosyltransferases
such as oligosaccharyltransferase,
and glutathionylspermidine synthetase/amidase.
Folic acid is a key vitamin in human
nutrition. Cellular folates contain a reduced pteridine
heterocycle and a poly-g-glutamyl peptide
"tail". Two enzymes, folypolyglutamate synthetase (FPGS)
and g-glutamyl hydrolase (GH), being
studied by our research group, catalyze the biosynthesis and
hydrolytic cleavage, respectively, of the polyglutamyl portion of
cellular folates and antifolate drugs. Recent research has
involved the synthesis of several fluoro- and phosphoamino acids and
their incorporation into folates and antifolate drugs. These
effortes have led to the synthesis of fluoropeptides or
phosphapeptides for biochemical investigation as inhibitors or
stimulators of the reactions catalyzed by FPGS or GH. In
collaborative research, our new compounds are beina used in intact
mammalian cells to assess the role of polyglutamate formation and
hydrolysis in normal folate-dependent one-carbon biochemistry and
also in the pharmacology of antifolate drugs used in the treatment
of cancer.
Trypanothione (TSH), found exclusively in trypanosomatid parasites,
is a derivative of the ubiquitous antioxidant, glutathione (GSH).
The biosynthesis of TSH involves bis-acylation of the polyamine,
spermidine, by GSH. The intermediate monoacylated GSH,
glutathionylspermidine (Gsp), but not TSH, is found in E. coli and
both its synthesis and hydrolysis are effected by a unique
bifunctional enzyme, Gsp synthetase/amidase. We have extended
the phophapeptide research mentioned above to the development of
new inhibitors of TSH biosynthesis as possible anti-trypanosomal
drugs. Based on an understanding of the mechanism of Gsp we
have designed and synthesized specific phophapeptide inhibitors of
the synthetase domain and, in collaborative research, have used
these compounds to study the regulation of catalysis by this
bifunctional enzyme.
Oligosaccharyltransferase (OST), an enzyme that catalyzes the N-glycosylation
of proteins, is a membrane-bound protein that we isolate from
yeast. Initial research has centered on the use of
synthetically accessible small substrates, characterization of the
chemical structure of the glycopeptide product, and the use of
isotopically labeled peptide substrates for mechanistic
studies. Recent research has focused on the use of larger
peptides and proteins as substrates and has led to extensive studies
with invertase-derived peptides of varying length. Kinetic
data combined with structure-activity data has allowed us to
develope a working hypothesis concerning peptide substrate
recognition by OST. Current research also involves the
synthesis of a variety of substrates and inhibitors designed to
answer specific mechanistic questions. As in the research with
the folate polyglutamates and trypanothione, the goal here is to
understand the mechanism of this reaction in sufficient detail that
it will be feasible to design and synthesize potent and specific
inhibitors for use in the study of cellular glycoprotein
biosynthesis.
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