The enzyme Myristoyl CoA-transferase (NMT) transfers myristate to an N terminal glycine of a target protein or peptide. Myristoylation may serve to anchor peptides or proteins in a hydrophobic region. The mechanism of action of NMT will be investigated. An important question is whether myristate forms a covalent intermediate with the enzyme. To answer this question [3H] myristoyl CoA will be added to NMT in the absence of peptide acceptor, or in the presence of a peptide analog which is not myristoylated. The enzyme will be degraded with trypsin and chymotrypsin. The resulting peptides will be separated and examined for radio-activity. If radiolabeled material is associated with one of the peptides, formation of a covalent enzyme-substrate complex probably occurred. Aza peptides are peptides in which the S1 amino acid is replaced by NH-NR-CONH. These peptides inhibit serine and thiol proteases. Inhibition is due to acylation of the active site -OH or -SH. The resulting acyl-enzyme hydrolyses slowly. The structure of the inactive enzyme will be determined by NMR and chemical approaches. Understanding of the mechanism will be important in improving these inhibitors. Aza-peptide inhibitors are attractive for pharmacological applications, since they are not subject to proteolytic digestion and are resistant to chemical degradation. They also are highly selective. CO formation occurs in organisms ranging from fungi to mammals. CO has biological effects similar to NO. It has been called a neuro transmitter. Little is known about the enzymatic mechanism of CO formation. The investigator and his colleagues have purified an enzyme from K. Pneumoniae which catalyzes the formation of CO. To ascertain the enzyme mechanism, non enzymatic model systems will be investigated. For instance: CH3-CO-CH2-CO-CH3 is non-enzymatically converted to CO and other products in the presence of H2O2. ESR experiments will be done to detect free radicals. It is anticipated that an understanding of the mechanism will enable the applicant to design an inhibitor which can be used to inhibit CO production in intact bacterial cells.
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