Mechanism of Catalysis and Inhibition of Mammalian Protein Farnesyltransferase.

Mammalian protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon prenyl group from farnesyl diphosphate (FPP) to a cysteine residue near the carboxyl terminus of many proteins, including several key molecules involved in signal transduction. Common substrates include oncogenic Ras proteins, and several FTase inhibitors are under development for the treatment of various cancers. FTase is a member of the newest class of zinc metalloenzymes that catalyze sulfur alkylation, and the work described here provides further insight into the mechanism of catalysis for this enzyme, which may lead to an increased understanding of the substrate specificity and inhibition of FTase. The reaction catalyzed by FTase results in two products: diphosphate and farnesylated protein or peptide. To measure the rate constant for diphosphate dissociation, a coupled fluorescent assay was developed. This assay can also be used to measure FTase activity for mechanistic studies and for high throughput screening to identify FTase substrates and inhibitors. The dissociation of the farnesylated product bound to FTase is accelerated by binding FPP. This step is crucial for substrate selectivity, as measured by substrate analog studies, and inhibition studies demonstrate that some FPP-competitive inhibitors function by slowing product dissociation. Together, these studies suggest that the binding of a second substrate molecule to facilitate product release is an important determinant of the substrate specificity, and potentially of the physiological regulation of FTase. To investigate the structure of the chemical transition state of FTase, the primary 14C and α-secondary 3H kinetic isotope effects (KIEs) were measured using transient kinetics. These data suggest that the FTase reaction proceeds via a concerted mechanism with dissociative character, facilitated by the zinc ion which coordinates the thiolate of the peptide substrate. The effects of the Mg2+ concentration and mutations of positively charged residues that interact with the diphosphate leaving group on the α-secondary KIE suggest that Mg2+ and these side chains both stabilize the transition state for farnesylation and facilitate a conformational rearrangement of bound FPP that occurs prior to farnesylation. Finally, the dependence of the α-secondary KIE on peptide structure indicates that this FPP conformational change is important for substrate specificity.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57672/2/jpais_1.pd

Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca₁a₂X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca₁a₂X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors

Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor

Pam18, the J-protein cochaperone of the Hsp70-based mitochondrial import motor, forms a heterodimer with the structurally related protein Pam16. Genetic and biochemical studies suggest a critical role of this interaction in maintaining Pam18's association with the translocon rather than its previously proposed regulatory role