STRUCTURE AND MECHANISM OF YEAST PYRUVATE DECARBOXYLASE

  • Jordan, Frank (PI)
  • Jordan, Frank (PI)
  • Jordan, Frank (PI)
  • Jordan, Frank (PI)

Project Details

Description

This proposal is concerned with delineation of remaining structural and
mechanistic questions on pyruvate decarboxylase (PDC, E.C. 4. 1. 1. 1),
perhaps the simplest nonoxidative decarboxylase requiring thiamin
diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance
in human metabolism). The brewer' yeast enzyme was recently purified as
fully active alpha4 and beta4 homotetramers and the alpha4 structure was
crystallized by the PI and collaborators at the VA Hospital in Pittsburgh
pointing to the likely success of resolution to at least 2.4 angstroms.
With the evidence already accumulated from the 3 angstroms map of the
enzyme, for the first time on this representative of a large group of
alpha-keto acid decarboxylases, rational experiments can be designed to
probe: a. the conformation of the enzyme-bound coenzyme in the absence of
substrate, as well as in the three covalent substrate-coenzyme complexes
invoked, based on chemical analogy; b. the chemical behavior in the
absence and presence of substrate of the two aromatic rings (thiazolium
and 4-aminopyrimidine), and test of the hypothesis that both rings ( not
only the thiazolium) are participants in catalysis; c. the environment of
the coenzyme and the function of amino acids surrounding it in catalysis;
and d. the effect of pyruvamide, a nondecarboxylatable substrate
surrogate that is capable of shifting the enzyme from a low to a high
activity form, on structure and mechanism. Most prominent among the
tools to address these questions will be : a. x-ray crystallographic
methods (this part of the research is a collaborative effort with Furey,
Sax and coworkers at the VA Hospital Biocrystallography Lab/ Univ. of
Pittsburgh) ; b. site-directed mutagenesis of amino acids found near the
catalytic and regulatory sites based on the x-ray crystallographic and
mechanistic information; c. further elucidation of the chemistry and
enzyme-bound environment of the enamine, one of the three ThDP-substrate
covalent complexes on PDC; d. multinuclear magnetic resonance on ThDP
bound to PDC, with ThDP labelled at the C2, Nl' and N4' atoms to provide
information concerning the state of ionization, and the charge densities
of the coenzyme during the catalytic sequence in the absence and in the
presence of regulators;. and 5. modeling based on the x-ray coordinates
to help refine mechanistic models, to help design even more insightful
experiments, and to help construct three dimensional models of two
enzymes with very high sequence homology to PDC's (pyruvate oxidase and
acetolactate synthetase) that have not been crystallized yet, and that
have identical mechanisms through pyruvate decarboxylation, but diverge
significantly thereafter. Undoubtedly, the fundamental questions to be
resolved by the proposed research, will be of profound interest and
significance to many other research groups working on thiamin around the
globe.
StatusFinished
Effective start/end date5/1/9311/30/14

Funding

  • National Institute of General Medical Sciences: $273,322.00
  • National Institute of General Medical Sciences: $210,098.00
  • National Institute of General Medical Sciences: $206,602.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $365,878.00
  • National Institute of General Medical Sciences: $265,396.00
  • National Institute of General Medical Sciences: $370,300.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $366,894.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $355,390.00
  • National Institute of General Medical Sciences: $268,800.00
  • National Institute of General Medical Sciences: $268,800.00
  • National Institute of General Medical Sciences

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Radiation
  • Spectroscopy
  • Catalysis
  • Cell Biology

Fingerprint

Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.