The objective of these studies on aconitase is to provide a detailed account of the sequence of conformational changes and proton transfers that occur after the reaction chemistry of the enzyme that are required before another substrate can be processed. This information will contribute to the long term goal of understanding the principles used in evolution to reverse the changes that occur in the active site of all enzymes during the catalytic process and show how an understanding of recycling provides insights into otherwise inaccessible aspects of the reaction itself. Recognition of recycling transitions and the ligands with which they react will specifically be essential for the design of drugs aimed at enzyme targets in vivo. A number of properties of the FeS-aconitase reaction suggest that a study of recycling will be informative by use of noncompetitive inhibitors of the enzyme. Slow recycling may be responsible for the unusually slow turnover of the enzyme, only 23 s(-1). The nature of slow recycling steps will be determined by use of buffers, D(2)O(2), viscogens and potential activators by examining their affects on the Kii and Kis values of specific inhibitors. From the large amount of high-resolution structural information available for aconitase there is nothing to suggest conformational variants with affinity for different ligands. However, it seems probable that the only species that has been studied so far is the non-specific isoform required in all recycling schemes. The kinetic demonstration of forms specific for citrate and for cis-aconitate may lead others to study the nature of the changes in conformation required for the reaction and the recycling process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM020940-35
Application #
6385282
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
1979-01-01
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2003-06-30
Support Year
35
Fiscal Year
2001
Total Cost
$7,887
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Rose, Irwin A; Weaver, Todd M (2004) The role of the allosteric B site in the fumarase reaction. Proc Natl Acad Sci U S A 101:3393-7
Rose, Irwin A; Nowick, James S (2002) Methylglyoxal synthetase, enol-pyruvaldehyde, glutathione and the glyoxalase system. J Am Chem Soc 124:13047-52
Rose, I A (1998) How fumarase recycles after the malate --> fumarate reaction. Insights into the reaction mechanism. Biochemistry 37:17651-8
Rose, I A (1997) Restructuring the active site of fumarase for the fumarate to malate reaction. Biochemistry 36:12346-54
Rose, I A (1995) Partition analysis: detecting enzyme reaction cycle intermediates. Methods Enzymol 249:315-40
Rose, I A; Warms, J V; Yuan, R G (1993) Role of conformational change in the fumarase reaction cycle. Biochemistry 32:8504-11
Seeholzer, S H (1993) Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity. Proc Natl Acad Sci U S A 90:1237-41
Rose, I A; Kuo, D J (1992) Role of CO2 in proton activation by histidine decarboxylase (pyruvoyl). Biochemistry 31:5887-92
Rose, I A; Warms, J V; Kuo, D J (1992) Proton transfer in catalysis by fumarase. Biochemistry 31:9993-9
Seeholzer, S H; Jaworowski, A; Rose, I A (1991) Enolpyruvate: chemical determination as a pyruvate kinase intermediate. Biochemistry 30:727-32

Showing the most recent 10 out of 22 publications