A general investigation of the mechanisms of catalytic hemoproteins and of the relationships between hemoprotein structure and activity is in progress. This investigation includes, but is not limited to, studies of cytochrome P450, horseradish peroxidase, chloroperoxidase, myoglobin, prostaglandin synthase, myeloperoxidase, nitric oxide sythnthase and heme oxygenase. Key elements in the study of these proteins is the analysis of product structure, stereochemistry and isotopic substitution. Mass spectrometry is one of the principal techniques utilized to elucidate these parameters. In addition to product studies, heavy use is made of mechanism-based and other irreversible inhibitors to characterize the active sites. These irreversible inhibitors alkylate either the heme group or the protein. Mass spectrometry is essential for identification of the structures of the heme adducts, and consequently for the use of mechanism-based inhibitors as structural probes. The identification of protein residues that are modified by the inhibitors that react with the protein skeleton is also currently being pursued as an additional tool for analysis of the active site structure. We propose to use mass spectrometry to achieve this goal. These investigations, as in the past, are expected to make major contributions to our understanding of the hemoprotein mechanisms, to the development of potentially useful hemoprotein inhibitors and to the general understanding of protein structure and function.
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