TRANSITION STATE ANALYSIS OF ENZYMATIC REACTIONS

Project: Research project

Project Details

Description

Enzymatic reactions involve the formation of one or more metastable
transition states on the pathway from substrates to products.
Transition states of enzymatic reactions are characterized by
tightly bound complexes which can be in equilibrium with the
substrate or may be committed to product formation. Recent
advances in the application of heavy-atom kinetic isotope effects
have permitted the modeling of transition state structures for
enzyme catalyzed reactions based on experimentally determined
kinetic isotope effects. The purpose of this research is to
determine transition state structures for purine nucleoside
phosphorylase and inosine hydrolase, enzymes which catalyze
phosphorolysis or hydrolysis of the H-glycosidic bond of purine
nucleosides. Deficiency of purine nucleoside phosphorylase results
in an immunodeficiency disorder in humans. Inosine hydrolase is
an enzyme of purine salvage found in tryanosomes but not in humans.
Trypanosomes have no de novo purine biosynthesis and thus depend
on salvage pathways. The characterization of the transition states
for these enzymes should prove useful in the development of
transition state analogues for these enzymes. The transition state structure for purine nucleoside phosphorylase
will be established from the family of kinetic isotope effects for
the arsenolysis reaction and for the reaction with poor substrates.
The transition state structure will be modeled into the crystal
structure recently established for purine human erythrocyte
nucleosidase. The atomic motions required to convert the reactant
enzyme-substrate complex into the transition state complex should
be evident from the two structures. A tightly bound intermediate
which is formed between purine and purine nucleoside phosphorylase
in the absence of phosphate will be characterized. Inosine hydrolase will be isolated from the trypanosome Crithidia
fasciculata. Following transition state analysis by heavy atom
kinetic isotope effects, the enzyme will be cloned from a genomic
DNA library from C. fasciculata library, sequenced and expressed
in E. coli. Attempts will be made to crystallize the protein for
the eventual goal of correlating the transition state structure and
the catalytic site structure.
StatusFinished
Effective start/end date12/31/897/31/21

Funding

  • National Institute of General Medical Sciences: $49,768.00
  • National Institute of General Medical Sciences: $576,150.00
  • National Institute of General Medical Sciences: $658,125.00
  • National Institute of General Medical Sciences: $615,685.00
  • National Institute of General Medical Sciences: $116,253.00
  • National Institute of General Medical Sciences: $231,420.00
  • National Institute of General Medical Sciences: $623,731.00
  • National Institute of General Medical Sciences: $686,855.00
  • National Institute of General Medical Sciences: $576,150.00
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences: $547,454.00
  • National Institute of General Medical Sciences: $665,271.00
  • National Institute of General Medical Sciences: $576,150.00
  • National Institute of General Medical Sciences: $628,305.00
  • National Institute of General Medical Sciences: $499,254.00
  • National Institute of General Medical Sciences: $344,730.00
  • National Institute of General Medical Sciences: $85,726.00

ASJC

  • Drug Discovery
  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Molecular Biology
  • Spectroscopy
  • Catalysis
  • Genetics
  • Radiation
  • Chemistry(all)
  • Cancer Research
  • Parasitology
  • Microbiology

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