Yeast protein farnesyltransferase: a pre-steady-state kinetic analysis.
Protein farnesyltransferase catalyzes alkylation of the cysteine in a carboxy-terminal CaaX motif where a is typically an aliphatic amino acid and X is alanine, methionine, serine, glutamine, or cysteine by a farnesyl residue. The modification enhances the lipophilicity of farnesylated proteins and promotes their association with membranes as part of their normal cellular function. Among the proteins modified by farnesyl residues is Ras, an important component in the signal transduction network for cell division that has been implicated in several forms of human cancer. In this paper, we describe isotope trapping, rapid quench, and single turnover experiments with the yeast enzyme using farnesyl diphosphate and the short peptide RTRCVIA as substrates. The kinetic constants for substrate binding, chemistry, and product release were determined from a fit of the differential equations describing the minimal catalytic mechanism to the kinetic data by numerical integration. Rate constants for chemistry and product release were 10.5 and 3.5 s(-1), respectively. The dissociation rate constant (33 s(-1)) for release of peptide from the ternary enzyme-substrate complex was three times larger than the rate constant for chemistry. The enthalpy of reaction, delta Hrxn = -17 +/- 1 kcal/mol for farnesylation of cysteine, was measured by microcalorimetry. Isotope trapping experiments revealed that the enzyme-farnesyl diphosphate complex was efficiently trapped by peptide but that the enzyme-peptide complex was not trapped by farnesyl diphosphate. These results are consistant with an ordered mechanism for formation of a catalytically competent ternary enzyme-farnesyl diphosphate-peptide complex.
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Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Poulter
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D019883:Alkyl and Aryl Transferases; D000478:Alkylation; D001665:Binding Sites; D002151:Calorimetry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism*
D014166:Transferases; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D003545:Cysteine