Structural biochemistry of the protein farnesyltransferase reaction path

The membrane localization and function of many intracellular signal transduction molecules require the covalent post-translational modification by specific lipids. Protein farnesyltransferase (FTase) catalyzes the functionally required attachment of a farnesyl lipid group to many essential signal transduction proteins, including members of the Ras superfamily of GTP-binding proteins. The farnesylation of Ras oncoproteins, which are associated with 30% of human cancers, is essential for their transforming activity. Inhibitors of FTase are currently in clinical trials for the treatment of cancer.;Biochemical experiments have elucidated many of the properties of the FTase reaction cycle. The crystal structure of FTase without bound substrate or product ligands provided a starting point to understand the mechanisms of this enzyme on a structural level. To understand these mechanisms further, I have determined the crystal structures of complexes of FTase with the farnesyl diphosphate (FPP) and peptide substrates, nonreactive substrate analogs, and farnesylated products. Together, these structures represent the known steps of the reaction cycle. From these observations, the determinants of substrate specificity, the basis for ordered substrate binding, the role of metal ions in the reaction, the nature of the transition state, and the structural basis for involvement of substrate binding in the release of product have been deduced. In addition, these complexes give insight into the mechanisms of prenylation catalyzed by two related enzymes, protein geranylgeranyltransferase type I and protein geranylgeranyltransferase type II.;I have also investigated the structural determinants for inhibition by two peptide-substrate competitive inhibitors that cause tumor regression in animal models. One of these inhibitors has entered in clinical trials for the treatment of human cancer. Complexes with these inhibitors represent the first crystal structures determined for human FTase. The structures presented in this dissertation not only increase our understanding of the mechanisms of protein prenylation but should also aid in the design and optimization of anti-cancer drugs.