Enzymology of DHHC‐mediated protein palmitoylation

Protein S ‐palmitoylation is a post‐translational modification in which a 16‐carbon fatty acid is thioester linked to a cysteine residue. It is mediated by a family of enzymes with a conserved Asp‐His‐His‐Cys sequence (DHHC). Incubation of DHHC proteins with palmitoyl‐Coenzyme A (palm‐CoA) both in the presence and absence of a protein substrate results in the DHHC protein itself being acylated. Enzyme autoacylation is a prerequisite for substrate palmitoylation. Using two approaches, we are testing the hypothesis that DHHC enzymes use a two‐step transfer mechanism where the autoacylated species represents an acyl‐enzyme intermediate. Mutation of the DHH C cysteine residue results in loss of both enzyme and substrate acylation. Enzyme acylation is hydroxylamine‐sensitive, suggesting palmitate is attached via a thioester linkage to cysteine. In our first approach to directly monitor transfer a DHHC protein is reacted with [ 3 H]palm‐CoA and then purified from any free [ 3 H]palm‐CoA. The charged [ 3 H]palm‐DHHC is then incubated with a protein substrate to determine if the radioactive acyl group is transferred. Our second approach is to use steady‐state kinetics to determine if DHHC proteins use a Bi‐Bi (ping‐pong) mechanism. A fluorescent peptide, HPLC‐based protein acyltransferase assay has been developed to achieve the higher substrate concentrations needed for kinetic analysis. Funding was provided by a pre‐doctoral fellowship from the American Heart Association (BCJ) and NIGMS (ML).