| Peptide deformylase catalyzes the specific cleavage of formyl moiety from newly synthesized proteins in bacteria. It is highly selective for N-formyl peptides over N-acetyl peptides. To probe the mechanism of this high selectivity, more fluoroacetyl peptides were synthesized and characterized using a continuous deformylase assay developed previously. Kinetic constants of the substrates suggest that both steric and electronic effects play a role. In addition, factors contributing to the low KM of N-formyl-Met-Leu-p-nitroanilide were investigated.; Deformylase in vivo is capable of deformylating most of the polypeptides in bacterial cells bearing diverse N-terminal sequences. To systematically examine the sequence specificity of peptide deformylase, a peptide library that contained all possible N-terminally formylated tetrapeptides had been constructed and screened in this lab. In this work, detailed kinetic studies were carried out with representative substrates selected from the library. The results confirmed that, although a broad-specificity enzyme, peptide deformylase cleaves formyl peptides at drastically different rates. It was found that formyl-Met-X-Z-Tyr (X = any amino acid except for aspartate and glumate; Z = lysine, arginine, tyrosine or phenylalanine) and formyl-Met-Phe-Tyr-(Phe/Tyr) peptides are optimal substrates.; Deformylation is a conserved and distinctive feature of protein biosynthesis in bacteria and deformylase is essential for bacterial survival. Its absence in eukaryotes makes it an attractive target for antibacterial chemotherapy. Rational design of deformylase inhibitors was undertaken by replacing scissile bond of high-affinity substrate with thiol, carboxylic and aldehyde functionalities. Competitive inhibitors were obtained among which the most potent one had a KI of 81 nM. To discover inhibitors with different structural features, a combinatorial library of deformylase inhibitors was constructed on solid phase by split-pool synthesis. The structures of most active inhibitors were deduced by deconvolution.; Alternative to inhibitor design, a novel approach to antibacterial drug design was developed, in which a pharmacologically inactive prodrug was selectively processed by the bacterial deformylase to release a cytotoxic drug inside the bacterial pathogen. The prodrugs were a series of 5'-dipeptidyl derivatives of 5-fluoro-2'-deoxyuridine (FdU). In the presence of peptide deformylase, the formyl group at the N-termini of the dipeptides is removed, leading to an intramolecular cyclization and release of FdU. The broad substrate specificity of peptide deformylase allows for the use of unnatural amino acids in the prodrugs, which have improved in vivo stability. These compounds should be biologically inactive because 5'-acylation blocks the 5'-phosphorylation of FdU, a necessary step for FdU to become an active antimetabolite. Because the active drug is produced only inside the bacterial pathogen, these prodrugs should have low cytotoxicity to the host. |
