Coenzymes play a critical role in many enzyme catalyzed processes by providing functionality not accessible in the naturally encoded amino acids. In this research, the potential utility of a series of catalytically competent coenzyme amino acid chimeras in protein design will be explored. These residues incorporate the core functionality of the coenzymes pyridoxal/pyridoxamine phosphate, nicotinamide adenine dinucleotide and thiamin diphosphate as integral constituents of alpha- amino acid constructs. In these forms, the coenzyme functionalities can be incorporated into a polypeptide at any position within the primary sequence via solid phase peptide synthesis. The long term goals of this research are to develop complex an appropriate peptidyl architecture to host the functional coenzyme moieties. In addressing these challenges knowledge will be gained on the manipulation of the peptide architecture for the construction of new catalysts.
The specific aims of this proposal center on the development of three new catalytic systems. (l) An alpha-amino acid/alpha-keto acid transaminase that incorporates a pyridoxamine amino acid chimera, (2) A thiazolium dependent alpha-keto acid decarboxylase and (3) A nicotinamide dependent system for the stereospecific reduction of unactivated ketones that employs divalent zinc for carbonyl activation. Each amino acid will be built into a polypeptide architecture of de novo design. The motifs that will be employed include alpha-helical bundles and beta-beta-alpha supersecondary structural elements. The complexity of these constructs will allow for significant structural variation necessary for developing the reactivity and selectivity that is the hallmark of enzyme catalysis. This research will provide important insight into the interplay between peptide structure and function in coenzyme catalysis. Studies of this kind may lead to the evolution of selective new catalysts for key organic transformations.
