Through the broad, long term objectives of this research application we seek to understand the molecular mechanisms of metalloenzyme function, with specific focus on the mechanisms by which biological macromolecules recognize other macromolecular and small organic structures. Systematic investigations are proposed that focus on three major classes of recognition events in biological systems. (1) Protein -Protein Recognition. Here we seek to define the role of specific surface interactions; electrostatic, hydrogen bonding, and hydrophobic free energies provided through surface complimentary, which define the specificity and affinity in the formation of complexes between the metalloproteins involved in electron transfer events in cytochrome P-450 dependent oxygenase catalysis. (2) Protein Small Molecule Recognition. In this Specific Aim we seek to ascertain how the same fundamental forces of electrostatics, hydrogen bonding and the hand - glove fit of a substrate into the active site of an enzyme can give rise to the observed high degree control of regio- and stereo- specificity in cytochrome P-450 catalysis. In this regard we must define the role of protein and substrate motion on the recognition event. (3) Protein - Nucleic Acid Recognition. Again the same fundamental forces control recognition processes, but in this case we focus on our exciting recent discovery of a role for solvent water in mediating hydrogen bond recognition between protein and nucleic acid components. Representative systems in the binding/catalytic class (restriction endonuclease, recombinase) and binding/non-catalytic class (repressor-operator) are proposed for investigation. The question of molecular recognition is a central paradigm of molecular biology, playing central roles in most, if not all, cellular processes. Failed recognition events have been implicated in numerous disease states ranging from flawed control of gene regulation and cellular proliferation, to defects in specific metabolic activities. Historically, questions of molecular recognition have been approached through organic synthesis through actual structural studies of biomolecular complexes. Our proposed research efforts in this competitive renewal make concerted use of broad interdisciplinary tools and techniques. In particular, the use of recombinant DNA technology in concert with advanced biophysical methods has proven to be ideal for understanding the fundamental mechanisms of recognition in metalloenzyme systems. Our thrust in biological oxidations utilizes as exemplary systems the cytochromes P-450 which play great significance in the biotransformations of hepatic and adrenal tissues of humans and a plethora of oxidative metabolism across the plant, animal and archaebacterial kingdoms. Thus, the central problems of metalloenzyme biology, chemistry, and biophysics will be attacked through the selective choice of the tractable systems proposed in this application, and the combined use of recombinant DNA technology and state of the art structure - function characterization in order to place molecular mechanisms on a firm foundation for further scientific development.
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