Volume 272, Number 1, Issue of January 13, 1997 pp. 20-23
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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COMMUNICATION:
Evidence for a Catalytic Role of Zinc in Protein Farnesyltransferase
SPECTROSCOPY OF Co²⁺-FARNESYLTRANSFERASE INDICATES METAL COORDINATION OF THE SUBSTRATE THIOLATE

(Received for publication, October 29, 1996, and in revised form, November 8, 1996)

Chih-Chin Huang , Patrick J. Casey ^§ and Carol A. Fierke

From the Departments of  Biochemistry and ^§ Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

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 (₃₂₀ = 1030 M¹ cm¹) consistent with the coordination of one cysteine side chain and also ligand field bands (₅₆₀ = 140 M¹ cm¹) indicative of a pentacoordinate or distorted tetrahedral metal geometry. Most importantly, the ligand-metal charge transfer band displays an increased intensity (₃₂₀ = 1830 M¹ cm¹) in the ternary complex of FTase·isoprenoid·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·FPP·peptide substrate complex and that the rate constant for the chemical step is 17 s¹. 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.

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