The long-term goal is to relate molecular structure to specificity and reactivity in enzymes, with high-resolution x-ray structure analysis as a primary method. This proposal focuses on local and global conformational changes that are essential in catalysis and control, using enzymes that provide paradigms for domain 'motions', for transition state stabilization, and for interactions mediated through proteins by structural changes. Some of the selected enzymes are dependent on metallo- or vitamin-based cofactors. Genetic engineering, spectroscopic, and biochemical techniques will also be deployed to show how the function of each protein depends on conformational equilibria. Descriptions of domain rearrangements will be obtained for two prototypical enzymes: B12-dependent methionine synthase (MetH) and the flavoenzyme, thioredoxin reductase (TrxR). MetH from E. coli is a modular 136 kDa protein with regions that bind homocysteine, methyltetrahydrofolate, B12, and adenosylmethionine (AdoMet). MetH operates by an alternating domain scheme that requires different modules to interact in turn with the B12 domain. Structure-function analyses will define the arrangements and interactions of modules in the catalytic and reactivation states, and will help to explain how conversions between conformations are triggered. Impairment of human methionine synthase, an analog of the E. coli enzyme, is responsible for many of the manifestations of B12 deficiency. TrxR, a two-domain protein, employs domain rotations to present NADPH and the active site disulfide alternately to the FAD cofactor. These rotations limit the rate of catalysis. One of the reactive conformations has been described by Kuriyan and coworkers. The other conformer will be analyzed, stabilizing it via -S-S- links to the substrate, thioredoxin. Conformation changes that occur in catalytic cycles or modulate activity in cytidylyltransferases and methylenetetrahydrofolate reductase (MTHFR) will be established from structures of substrate, product and intermediate complexes. Initial analyses of glycerol-3-phosphate:cytidylyltransferase have revealed local conformational changes linked to substrate binding, and imply interactions between the active sites of the dimeric molecule. Structures of intermediates, including a transition state mimic, will be determined. E. coli MTHFR is a bacterial model for investigation of the role of this enzyme in controlling levels of homocysteine, a known risk factor for cardiovascular disease. Objectives are analyses of structures of wild type and mutant enzymes with folates bound, and the crystallization of human MTHFR, which displays allosteric control by AdoMet.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysical Chemistry Study Section (BBCB)
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Flicker, Paula F
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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