title: Evidence for a catalytic role of zinc in protein farnesyltransferase. Spectroscopy of Co2+-farnesyltransferase indicates metal coordination of the substrate thiolate.
abstract: Protein farnesyltransferase (FTase) is a zinc metalloenzyme that catalyzes the addition of a farnesyl isoprenoid 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 substitution of cobalt for zinc. The absorption spectrum of Co2+-FTase displays a thiolate-Co2+ charge transfer band (epsilon320 = 1030 M(-1) cm(-1)) consistent with the coordination of one cysteine side chain and also ligand field bands (epsilon560 = 140 M(-1) cm(-1)) indicative of a pentacoordinate or distorted tetrahedral metal geometry. Most importantly, the ligand-metal charge transfer band displays an increased intensity (epsilon320 = 1830 M(-1) cm(-1)) in the ternary complex of FTase x isoprenoid x peptide substrate indicative of the formation of a second Co2+-thiolate bond as cobalt coordinates the thiolate of the peptide substrate. 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 x FPP x peptide substrate complex and that the rate constant for the chemical step is 17 s(-1). These data provide evidence that the zinc ion plays an important catalytic role in FTase, most likely by activation of the cysteine thiol of the protein substrate for nucleophilic attack on the isoprenoid.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: C C
authors.initials: CC
authors.lastname: Huang
authors.forename: P J
authors.initials: PJ
authors.lastname: Casey
authors.forename: C A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D019883:Alkyl and Aryl Transferases; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D003035:Cobalt; D003545:Cysteine / Q000737:chemistry; D051231:Farnesyltranstransferase; D051381:Rats; D011994:Recombinant Proteins; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D014166:Transferases / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry*
chemical_list: D001059:Apoproteins; D011994:Recombinant Proteins; D003035:Cobalt; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D015032:Zinc; D003545:Cysteine