| The enzymes protein farnesyltransferase (FTase) and methionine synthase (both MetH and MetE) are examples of a growing class of alkyl-transfer enzymes that have a catalytic zinc ion. Because Zn(II) has a d10 electron configuration, it is often replaced with Co(II) for spectroscopic studies. The electronic properties of Co(II) make it ideally suited for further spectroscopic investigation of the active site of these enzymes. Although the Co(II)-substituted active site is generally assumed to be isostructural with the native Zn(II) active site, this assumption has not often been tested. This dissertation described an extended X-ray absorption fine structure (EXAFS) comparison of the structures of the Zn(II) and Co(II) active sites in these enzymes in the resting state, in the substrate-bound structure, and in the product bound form. Cobalt EXAFS data reveal structural differences between the Co(II) and Zn(II) forms, but confirm earlier Zn-EXAFS data showing that the substrate thiolate, but not the product thioether, bind directly to the metal. X-ray absorption near edge structure (XANES) data for cobalt suggest that the cobalt site geometry is perturbed by the presence of the product even though it is not directly coordinated to cobalt. ENDOR results for FTase show exchangeable protons associated with the paramagnetic Co(II); however, the estimated Co-O bond length is ∼ 2.7 A, much too long to be a coordinated ligand. These results suggest that the Co(II) site, and by analogy the Zn(II) site, is four coordinate, with ligation by a cysteine, a histidine, and a bidentate aspartate.;In addition, this dissertation describes studies of substrate specificity in FTase. There are ∼ 620 proteins in the human genome that contain a conserved cysteine residue fourth from the C-terminus that have unknown prenylation status. Of the remaining ∼ 400 proteins that were not studied in a previous library, a second library of 90 peptides based on proteins with unknown prenylation status was studied and 29 novel FTase substrates were identified to be farnesylated by FTase under multiple turnover conditions. |
