We will continue to examine the nature of enzyme reaction mechanisms by use of reaction intermediates that are generated either in situ as for enolpyruvate, or in a single step by inactivation of a functioning enzyme as in the case of triose-P isomerase and metal dependent FDP aldolase. Other enzymes that we will examine are methylglyoxal synthase, which may produce the same intermediate as triose-P isomerase, and 6-P-gluconate dehydrogenase, which may produce the same enediol as D-pentose-P isomerase. By studying the pH dependence for reaction of enzyme with the intermediate it is possible to determine the contribution of diol and diolate forms to the reaction. The maximum rate of conversion of the proposed intermediate to normal products will be compared with normal Vmax of catalysis by the enzymes to establish the identity to normal intermediate. Other methods will be explored to determine the amount and chemical nature of intermediates that are too labile to stand by these methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM020940-29
Application #
3484373
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-01-01
Project End
1993-02-28
Budget Start
1992-03-01
Budget End
1993-02-28
Support Year
29
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Institute for Cancer Research
Department
Type
DUNS #
872612445
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
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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