Identification of a Cysteine Residue Essential for Activity of Protein Farnesyltransferase

Abstract

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zincdepleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [ 3 H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys 299 , that is in the ␤ subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys 299 in the ␤ subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.

Probes of the Catalytic Site of Cysteine Dioxygenase

“…This possibility is not consistent with the fact that CDO activity is unaffected by thiol-modifying agents. Recombinant CDO was treated with iodoacetamide, a thiol-modifying reagent used in a prior investigation of residual thiols that are critical for enzyme function (25). After treating holo-CDO with iodoacetamide followed by its removal by ultrafiltration, enzyme activity was unaffected.…”

Substrate Binding Is Required for Release of Product from Mammalian Protein Farnesyltransferase

“…The eluates were removed to a new tube containing SDS sample buffer, and the proteins were resolved on a 14% SDS-PAGE gel, followed by transfer to a polyvinylidene difluoride filter. These samples were then subjected to immunoblot using anti-FTase or anti-Ras antiserum (31). Visualization was by the alkaline phosphatase method using a commercial kit (Promega).…”

Substrate specificity determinants in the farnesyltransferase β-subunit

“…The structure of rat farnesyltransferase reveals a hydrophilic cleft and a hydrophobic cleft, which have been proposed to bind the CaaX protein and the farnesyl diphosphate substrates, respectively (36). Cysteine-299 of the farnesyltransferase ␤-subunit, which is coordinated with a zinc ion (36) and has been proposed to be directly involved in catalysis (31,37,38), resides at the junction of these two clefts. The ␣ carbon of glycine-142 of the rat farnesyltransferase ␤-subunit, which corresponds to glycine-149 of the yeast farnesyltransferase ␤-subunit, is located approximately 2 nm from the ␣ carbon of cysteine-299.…”

“…The recently published structure of rat farnesyltransferase reveals a hydrophilic cleft at the junction of the ␣-and ␤-subunits, which has been proposed to bind the CaaX protein, and a hydrophobic cleft within the ␣-␣ barrel structure of the ␤-subunit, which has been proposed to bind the farnesyl diphosphate substrate (36). Previous studies had strongly implicated cysteine-299 of the ␤-subunit in both zinc binding and catalysis (31,37,38), and the structural data demonstrate that cysteine-299 coordinates with a zinc ion and resides at the junction of the hydrophobic and hydrophilic clefts (36). However, the currently available data do not provide a definitive view of protein or lipid substrate binding or the mechanism of catalysis.…”

“…Genetic (28)(29)(30)(31)(32) and biochemical (33)(34)(35) studies have provided evidence that both the ␣-and ␤-subunits are involved in protein substrate binding and that the active site is likely to be at the interface of the two subunits. The recently published structure of rat farnesyltransferase reveals a hydrophilic cleft at the junction of the ␣-and ␤-subunits, which has been proposed to bind the CaaX protein, and a hydrophobic cleft within the ␣-␣ barrel structure of the ␤-subunit, which has been proposed to bind the farnesyl diphosphate substrate (36).…”