Effect of loop variants on enzymatic activity in acyl‐protein thioesterases

Abstract

Acyl‐protein thioesterases (APT) depalmitoylate, or remove fatty acid modifications, from proteins attached to membranes. In humans, APT1 and APT2 are known to control the palmitoylation of Ras, a signaling oncogene whose mutations are linked to cancer. My goal was to analyze the relative importance of specific loop residues hypothesized to regulate the membrane binding and catalytic activity of APT1. To accomplish this goal, wild‐type and loop variants of APT1 were expressed in Escherichia coli and purified to homogeneity. Purified APT proteins were then assayed for changes in thermal stability to confirm that mutations did not significantly shift the folding of the APT. Changes in enzyme function were measured using fluorescent kinetic measurements on substrates with various alkyl chain lengths. From each kinetic assay, a Michaelis‐Menten plot was created and the catalytic constant (kcat) and catalytic efficiency (kcat/KM) were recorded. Multiple APT loop variants have been successfully engineered and purified. Enzyme activity results indicate that substrates with smaller methyl chains are hydrolyzed more quickly by wild‐type APTs while APT1 variant W71A showed hinderance of enzymatic function in these substrates. In the future, we plan to analyze a wider library of APT1 and APT2 variants for shifts in biological activity of APT upon loop substitution.