This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This subproject involves the structural biology of two important areas of basic biochemistry and medicine, (1) oxidation-reduction within flavor- or quino-enzymes and electron transfer to their redox partners and (2) the allosteric regulation of thrombin, a key member of the family of serine proteases involved in the blood clotting cascade. One aspect of the redox studies involves the interactions of a copper protein, amicyanin, with a tryptophylquinone-containing enzyme, methylamine dehydrogenase and a cytochrome. Several mutants of amicyanin have been prepared that display a range of effects on the rates and mechanism of electron transfer. Atomic resolution studies of these mutants under a variety of conditions are beginning to reveal the subtle changes produced by these mutations and how they effect this fundamental process. In a related area, two forms of the bacterial flavoenzyme sarcosine oxidase are under investigation, a simple monomeric form containing FAD only and a complex heterotetrameric form containing FAD, FMN and NAD Structures of several mutants of both enzyme, in complex with a variety of exogenous ligands, are currently being investigated to define the catalytic mechanism of substrate oxidation and the factors controlling the transfer of electrons, protons and labile intermediates between catalytic sites in the latter enzyme. Thrombin plays both a procoagulant role in blood clotting upon cleavage of fibrinogen and also an anticoagulant role in the cleavage of protein C. Regulation of these roles is allosterically mediated by the binding level of monovalent cations, particularly sodium. Alanine-scanning mutagenesis has identified over a dozen residues that strongly affect this sodium regulation. Structural studies of these mutant protein in the presence and absence of these various cations are currently underway. This work is designed to identify the rolls played by each of these residues in the allosteric regulation and the interplay between them.
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