Future research will expand on two questions raised by previous work. The process by which enzymes liberate their products into the medium, rarely accessible to investigation, has become the central issue of our ongoing kinetic studies of the fumarase reaction. The active site (A-site) of the enzyme sits at the bottom of a well from which the product, fumarate, F, must escape to complete the reaction. Recent structural studies identified a second molecule of substrate, situated at the top of the well, the B-site. The role of the B-site is unknown but has been used to explain the activation observed with high concentrations of substrate, malate. Our studies with a B-site mutant suggest that product release to the medium occurs via the B-site. The A-site is then available to substrate again and helps the product leave the B-site.II. Previous work has suggested that the use of glutathione in the glyoxalase enzyme system may have evolved by virtue of its role in activating a prior chemical step. This suggested to us that cells that do not contain glutathione such as mycobacteria could employ their major thiol, mycothiol, instead of GSH for the nonenzymatic step and lead to an MSH based glyoxalase system. We will establish the chemistry of the glyoxalase system of Mycobacter smegmatis. Since methylglyoxal is toxic to cells and must be removed by an active glyoxalse system, inhibition of a MSH based system could result in a treatment for many mycobacterial-based diseases such as tuberculosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM020940-36
Application #
6679525
Study Section
Biochemistry Study Section (BIO)
Program Officer
Ikeda, Richard A
Project Start
1979-01-01
Project End
2006-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
36
Fiscal Year
2003
Total Cost
$7,575
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
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

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