Mutational analysis of Saccharomyces cerevisiae Erf2‐Erf4 palmitoyl transferase reveals a two‐step reaction mechanism for protein palmitoylation by DHHC enzymes

DHHC protein acyl transferases (PATs) catalyze the palmitoylation of proteins through a two‐step mechanism that consists of autopalmitoylation of enzyme, using palmitoyl‐CoA, to create a palmitoyl‐enzyme intermediate followed by the transfer of the palmitoyl moiety to the protein substrate. Using the yeast Erf2‐Erf4 Ras PAT as a model, we have combined genetic and biochemical analysis to interrogate the molecular mechanism of Ras palmitoylation. Mutational analysis of Erf2 and Erf4 has allowed us to glean insights into the PAT catalytic mechanism. The Erf2 subunit contains the DHHC motif that comprises the catalytic core of the enzyme. A model is proposed in which the His adjacent to the Asp of the DHHC, His‐201, is required to deprotonate Cys‐203 to create the critical nucleophile for the reaction with palmitoyl‐CoA to create the acyl‐enzyme intermediate. His‐201 also appears to play a role in activation of the protein substrate thiol to create the palmitoylate protein product. The function of Erf4 is less clear. Deletion of ERF4 destabilizes Erf2 through an ubiquitin‐mediated pathway. A genetic screen for mutant ERF4 alleles has begun to elucidate a role for Erf4 in the palmitoylation reaction. Results of these studies and efforts to determine a structure of the DHHC domain will be discussed. This work is supported by NIH grants CA50211 and GM73976.