MECHANISM OF FLAVOPROTEIN REDUCTASES

Project: Research project

Project Details

Description

The determination of the relationship between an enzymes' three-
dimensional structure and its catalytic function is the long-range
objective of this research. We have selected, as appropriate system for
such investigations, a number of enzymes in the flavoprotein reductase
family, which contain some of the most mechanistically intriguing and
physiologically important enzymes found in nature. They serve key roles
in oxidative stress management in humans and bacteria (erythrocyte
glutathione reductase and macrophage alkyl hydroperoxide reductase, NADH
peroxidase), and in trypanosomes (trypanothione reductase). They serve
to detoxify heavy metals (mercuric reductase) and in mammalian liver to
provide substrates for xenobiotic removal (glutathione reductase). They
play key roles in carbohydrate metabolism (lipoamide dehydrogenase) and
have been suggested to participate in the proper folding of proteins via
catalysis of disulfide bond formation and rearrangement (thioredoxin
reductase and the E. coli dsbA gene product). The amino acid sequences
of all of these proteins have been determined, and the high resolution
three dimensional structures of six members of the family, including all
the enzymes under investigation, have been reported. Evidence has been
obtained supporting a common rate-limiting proton transfer occurring in
of the oxidative half-reactions for four of these enzymes. The
transition state for hydride transfer between reduced pyridine nucleotide
and flavin has been determined for glutathione reductase, and a mechanism
for enzymatic transition state stabilization has been proposed based on
our studies and the structure of the enzyme-nucleotide binary complex.
the extension of these studies with human erythrocyte glutathione
reductase and trypanothione reductase from the parasitic protozoan,
Trypanosoma congolense, will include the testing of our hypothesis using
a combination of kinetic, isotopic, and mutagenic approaches. In both
these systems, the involvement of an ion pair between a glutamate
carboxyl and a lysine amino group in hydride ion stabilization will be
assessed by mutagenesis of the glutamate to a glutamine or aspartate
residue. Structural and isotopic investigations of the most
mechanistically unique, and enigmatic, of the reductases, NADH
peroxidase, will be continued. The enzymes discussed represent ideal
subjects for the detailed structure/function investigations proposed in
this grant due to the availability of cloned, sequenced and overexpressed
genes, high resolution three-dimensional structures, and favorable
spectroscopic and isotopic properties. These studies will provide a
detailed description of the molecular forces that are utilized by these
enzymes to stabilize the transition state for hydride transfer, one of
the simplest, but most central, chemical redox reactions in biology.
StatusFinished
Effective start/end date2/1/931/31/94

Funding

  • National Institute of General Medical Sciences

ASJC

  • Catalysis

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