Measuring the activity of farnesyltransferase by capillary electrophoresis with laser-induced fluorescence detection.
Enzymatic farnesylation of oncogenic forms of Ras proteins is the initial step in a series of posttranslational modifications essential for Ras activity. The modification is catalyzed by the enzyme, protein farnesyltransferase (PFTase), which transfers a farnesyl moiety from farnesyl diphosphate to the protein. We employed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection to develop a rapid and sensitive method for the determination of PFTase activity in vitro. The limited substrate specificity of PFTase allowed us to use a fluorescently labeled pentapeptide instead of a Ras protein as a substrate for the enzyme; the product of the enzymatic reaction was the farnesylated pentapeptide. The product was separated from the substrate by CE and quantified with LIF detection. Under optimal conditions, the separation was achieved within 10 min with a resolution of 86. The mass and concentration limits of detection for the farnesylated product were 10(-19) mol and 0.28 nM, respectively. By measuring the rate of accumulation of the farnesylated product, we were able to determine the kinetic parameters of the enzymatic reaction. For yeast PFTase as an enzyme and difluorocarboxyfluorescein-labeled GCVIA peptide as a substrate, the values of k(cat) and K(M) were found to be (3.1 +/- 0.3)x10(-3) s(-1) and (12.0 +/- 1.2) nuM, respectively. Our results suggest that CE-LIF can be efficiently used for the determination of enzymatic activity of PFTase in vitro. After minor modifications, the developed method can be also applied to other reactions of enzymatic prenylation of proteins.
Berezovski
Maxim
M
Department of Chemistry, York University, Toronto, Ontario, Canada.
Li
Wei-Ping
WP
Poulter
C Dale
CD
Krylov
Sergey N
SN
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D051231:Farnesyltranstransferase; D005453:Fluorescence; D007700:Kinetics; D007834:Lasers; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D002021:Buffers; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase