Farnesyl protein transferase: Identification of K164α and Y300β as catalytic residues by mutagenesis and kinetic studies

Zhen Wu, Mark Demma, Corey L. Strickland, Evette S. Radisky, C. Dale Poulter, Hung V. Le, William T. Windsor

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

Farnesyl protein transferase (FPT) is an α/β heterodimeric zinc enzyme that catalyzes posttranslational farnesylation of many key cellular regulatory proteins, including oncogenic Ras. On the basis of the recently reported crystal structure of FPT complexed with a CVIM peptide and α-hydroxy-farnesylphosphonic acid, site-directed mutagenesis of the FPT active site was performed so key residues that are responsible for substrate binding and catalysis could be identified. Eight single mutants, including K164Nα, Y166Fα, Y166Aα, Y200Fα, H201Aα, H248Aβ, Y300Fβ, and Y361Fβ, and a double mutant, H248Aβ/Y300Fβ, were prepared. Steady-state kinetic analysis along with structural evidence indicated that residues Y200α, H201α, H248β, and Y361β are mainly involved in substrate binding. In addition, biochemical results confirm structural observations which show that residue Y166α plays a key role in stabilizing the active site conformation of several FPT residues through cation - π interactions. Two mutants, K164Nα and Y300Fβ, have moderately decreased catalytic constants (k(cat)). Pre-steady-state kinetic analysis of these mutants from rapid quench experiments showed that the chemical step rate constant was reduced by 41- and 30- fold, respectively. The product-releasing rate for each dropped approximately 10-fold. In pH-dependent kinetic studies, Y300Fβ was observed to have both acidic and basic pK(a) values shifted 1 log unit from those of the wild-type enzyme, consistent with a possible role for Y300β as an acid-base catalyst. K164Nα had a pK(a) shift from 6.0 to 5.3, which suggests it may function as a general acid. On the basis of these results along with structural evidence, a possible FPT reaction mechanism is proposed with both Y300β and K164α playing key catalytic roles in enhancing the reactivity of the farnesyl diphosphate leaving group.

Original languageEnglish (US)
Pages (from-to)11239-11249
Number of pages11
JournalBiochemistry
Volume38
Issue number35
DOIs
StatePublished - Aug 31 1999

ASJC Scopus subject areas

  • Biochemistry

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