Transcarboxylase (TC) is a large multi-enzyme complex that catalyzes the transfer of CO2 from methyl malonyl co-enzyme A to pyruvate to form oxaloacetate and propionyl co-enzyme A. The broad objectives of the proposal are to provide structural information for TC in solution and detailed information on the two classes of carboxylation sites found in separate subunits. Since these two carboxyl transfer half reactions and chemically similar to those for biomedically important (de)carboxylase enzymes, such as propionyl-CoA carboxylase and pyruvate carboxylase, knowledge of mechanism for the transcarboxylase active sites will be of value in structure-function analyses of carboxylases implicated in disease states. Transcarboxylase consists of three different kinds of subunit (12S, 5S and 1.3S) and thirty polypeptide chains with an overall molecular weight of 1.2 million Daltons. Since the subunits have been cloned, and can be reassembled, and since stable substrate-subunit complexes can be f ormed, TC offers a rare opportunity to study the structure, assembly and function of a large oligomeric enzyme complex. To exploit this important opportunity the proposal sets three main goals: 1. To probe the chemistry and mechanism of the 12S active site. Raman difference spectroscopy, principally with single crystals of 12S, will be used to define changes in substrate and active site groups upon substrate binding. 2. To probe the chemistry and mechanism of the 5S and 6S active sites. Raman difference spectroscopy will be used to probe the details of substrate-5S interactions, with studies involving single crystals of 5S playing a major role. 3. To map inter-subunit and active site-active site distances in holo TC using fluorescence resonance energy transfer.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK053053-09
Application #
7087860
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Sechi, Salvatore
Project Start
1997-08-15
Project End
2007-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
9
Fiscal Year
2006
Total Cost
$254,548
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Carey, Paul R (2006) Spectroscopic characterization of distortion in enzyme complexes. Chem Rev 106:3043-54
Bethel, Christopher R; Hujer, Andrea M; Hujer, Kristine M et al. (2006) Role of Asp104 in the SHV beta-lactamase. Antimicrob Agents Chemother 50:4124-31
Carey, Paul R (2006) Raman crystallography and other biochemical applications of Raman microscopy. Annu Rev Phys Chem 57:527-54
Zheng, Run; Zheng, Xiaojing; Dong, Jian et al. (2004) Proteins can convert to beta-sheet in single crystals. Protein Sci 13:1288-94
Hall, Pamela R; Zheng, Run; Antony, Lizamma et al. (2004) Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit. EMBO J 23:3621-31
Carey, Paul R; Dong, Jian (2004) Following ligand binding and ligand reactions in proteins via Raman crystallography. Biochemistry 43:8885-93
Hall, Pamela R; Zheng, Run; Pusztai-Carey, Marianne et al. (2004) Expression and crystallization of several forms of the Propionibacterium shermanii transcarboxylase 5S subunit. Acta Crystallogr D Biol Crystallogr 60:521-3
Carey, Paul R; Sonnichsen, Frank D; Yee, Vivien C (2004) Transcarboxylase: one of nature's early nanomachines. IUBMB Life 56:575-83
Zheng, Xiaojing; Rivera-Hainaj, Rosa E; Zheng, Yuangang et al. (2002) Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence. Biochemistry 41:10741-6
Wang, Y F; Hyatt , D C; Rivera, R E et al. (2001) Crystallization and preliminary X-ray analysis of the 12S central subunit of transcarboxylase from Propionibacterium shermanii. Acta Crystallogr D Biol Crystallogr 57:266-8

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