Evidence for a Catalytic Role of Zinc in Protein Farnesyltransferase

Abstract

Protein farnesyltransferase (FTase) is a zinc met- alloenzyme that catalyzes the addition of a farnesyl iso- prenoid to a conserved cysteine in peptide or protein substrates. We have substituted the essential Zn2? in FTase with Co2? to investigate the function of the metal polyhedron using optical absorption spectroscopy. The catalytic activity of FTase is unchanged by the substitu- tion of cobalt for zinc. The absorption spectrum of Co2?- FTase displays a thiolate-Co2? charge transfer band ? ( 320 ? 1030 M?1 cm?1) consistent with the coordination of one cysteine side chain and also ligand field bands ? ( 560 ? 140 M?1 cm?1) indicative of a pentacoordinate or distorted tetrahedral metal geometry. Most impor- tantly, the ligand-metal charge transfer band displays an increased intensity ( ? 320 ? 1830 M?1 cm?1) in the ter- nary complex of FTase?isoprenoid?peptide substrate in- dicative of the formation of a second Co2?-thiolate bond as cobalt coordinates the thiolate of the peptide sub- strate. A similar increase in the ligand-metal charge transfer band in a product complex indicates that the sulfur atom of the farnesylated peptide also coordinates the metal. Transient kinetics demonstrate that thiolate- cobalt metal coordination also occurs in an active FTase?FPP?peptide substrate complex and that the rate constant for the chemical step is 17 s?1. These data pro- vide evidence that the zinc ion plays an important cat- alytic role in FTase, most likely by activation of the cysteine thiol of the protein substrate for nucleophilic attack on the isoprenoid.