The overall objective of this research is to enhance our understanding of structure-function relationships, including the mechanism and regulation of the El component of the pyruvate dehydrogenase multienzyme complex isolated from E. coli. This enzyme requires thiamin diphosphate (the vitamin B coenzyme) and decarboxylates pyruvate, the product of glycolysis, to initiate a series of reactions, a major product being acetyl-Coenzyme A, the starting material for the citric acid cycle. Among the specific goals for the requested period are: (1) Production of wild-type and variant El enzymes (the variants being made by site directed mutagenesis of the plasmid specifically bearing only the gene coding for El) for both mechanistic and structural studies, including, determination of the X-ray structure in a collaboration wit William Furey, University of Pittsburgh (with whom the principal investigator has collaborated in the solution of the simpler yeast pyruvate decarboxylase structure); (2) Experiments designed to a. identify the amino acids responsible for catalysis, and assign the function in catalysis; b. study the fate of key thiamin-bound intermediates on the enzyme; c. study the rate-limiting steps in catalysis and the structure of transition states in wild-type and active center variant enzymes; d. identify the site of, and solve the mechanism of inactivation of El by substrate fluoropyruvate, 2-oxo-3-butynoic acid (a potent substrate analog inhibitor developed in the principal investigator's laboratory), a putative transition-state analog, and several highly inhibitory monoclonal antibodies (also developed in the principal investigator's laboratory); (3) Experiments to identify the site(s) at which a variety of metabolic regulators interact with the El, including the cofactors and the products of the reaction, as well as GTP- and the pathway by which the information i transmitted to the active center; and (4) Explore the principal investigator's hypothesis concerning the key reductive acetyl transfer between the El and E2 enzymes. Since this enzyme is at a key metabolic junction, the principal investigator believes that in the coming years he and his collaborators have an opportunity to contribute to a better molecular-level understanding of this member of a very large class of related multienzyme complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062330-03
Application #
6628939
Study Section
Biochemistry Study Section (BIO)
Program Officer
Preusch, Peter C
Project Start
2001-02-01
Project End
2005-01-31
Budget Start
2003-02-01
Budget End
2004-01-31
Support Year
3
Fiscal Year
2003
Total Cost
$343,688
Indirect Cost
Name
Rutgers University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
130029205
City
Newark
State
NJ
Country
United States
Zip Code
07102
Jordan, Frank; Arjunan, Palaniappa; Kale, Sachin et al. (2009) Multiple roles of mobile active center loops in the E1 component of the Escherichia coli pyruvate dehydrogenase complex - Linkage of protein dynamics to catalysis. J Mol Catal B Enzym 61:14-22
Nemeria, Natalia; Chakraborty, Sumit; Baykal, Ahmet et al. (2007) The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes. Proc Natl Acad Sci U S A 104:78-82
Chandrasekhar, Krishnamoorthy; Arjunan, Palaniappa; Sax, Martin et al. (2006) Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution. Acta Crystallogr D Biol Crystallogr 62:1382-6
Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew et al. (2006) A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct. J Biol Chem 281:15296-303
Baykal, Ahmet; Chakraborty, Sumit; Dodoo, Afua et al. (2006) Synthesis with good enantiomeric excess of both enantiomers of alpha-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases. Bioorg Chem 34:380-93
Baykal, Ahmet T; Kakalis, Lazaros; Jordan, Frank (2006) Electronic and nuclear magnetic resonance spectroscopic features of the 1',4'-iminopyrimidine tautomeric form of thiamin diphosphate, a novel intermediate on enzymes requiring this coenzyme. Biochemistry 45:7522-8
Jordan, Frank; Nemeria, Natalia S; Sergienko, Eduard (2005) Multiple modes of active center communication in thiamin diphosphate-dependent enzymes. Acc Chem Res 38:755-63
Nemeria, Natalia; Tittmann, Kai; Joseph, Ebenezer et al. (2005) Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity. J Biol Chem 280:21473-82
Zhang, Sheng; Zhou, Leon; Nemeria, Natalia et al. (2005) Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment. Biochemistry 44:2237-43
Jordan, Frank; Nemeria, Natalia S (2005) Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations. Bioorg Chem 33:190-215

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