MECHANISM OF ACTION OF FLAVOPROTEIN REDUCTASES

Project: Research project

Project Details

Description

The goal of the present application is to determine the precise
chemical mechanisms and transition state structures of the two
half-reactions catalyzed by glutathione reductase and NADH
peroxidase. In the case of glutathione reductase, we are
interested in determining the factors which result in the
differential transition state stabilization of the reductive half
reaction by the yeast, spinach and human erythrocyte enzymes.
The structure of human erythrocyte glutathione reductase has
been determined, and we will interpret the data to be obtained
within a precisely defined structural framework. We also propose
to analyze the rate-limiting nature and transition state structure
of proton transfers occurring in the oxidative half-reaction
catalyzed by glutathione reductase. For NADH peroxidase, we
propose to examine the chemical mechanism of peroxide
cleavage, and investigate a unique, non-redox role for the bound
cofactor, FAD. The methods that will be used to probe these
questions include: determination of steady-state kinetic
parameters for various nucleotide and reducible substrates,
analysis of steady-state and pre-steady-state primary and
secondary deuterium and tritium kinetic isotope effects,
determination of solvent equilibrium and kinetic isotope effects,
determination of enzyme-bound flavin and disulfide-dithiol redox
potentials, and stopped-flow identification of flavin
intermediates. The methodologies developed and information
obtained for glutathione reductase and NADH peroxidase will be
applicable to other flavoprotein reductases. Six enzymes may be
considered to be members of this family: glutathione reductase
trypanathione reductase, thioredoxin reductase, mercuric
reductase, lipoamide dehydrogenase and NADH peroxidase. All
these enzymes serve physiologically important roles, including
maintenance of intracellular thiol redox poise, providing reducing
equivalents for deoxyribonucleoside synthesis, heavy metal
detoxication, and removal of injurious peroxides. While they each
exhibit non-overlapping specificity for their respective reducible
substrates, they share an impressive and extensive number of
kinetic, stereochemical and physiocochemical similarities,
including a high degree of primary sequence homology, especially
int he active site region. A long term objective is to understand
the structural basis for the functional differences observed within
this family.
StatusFinished
Effective start/end date12/31/891/31/06

Funding

  • National Institute of General Medical Sciences: $375,750.00
  • National Institute of General Medical Sciences: $376,875.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $375,750.00

ASJC

  • Catalysis
  • Genetics
  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physiology
  • Microbiology

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