Investigating the catalytic mechanism of the yeast palmitoyltransferase Akr1p

Abstract

Protein palmitoylation is a widespread posttranslational modification in which cysteine thiols on a substrate protein are modified with a palmitoyl group. Defining the active site and catalytic mechanism of palmitoyltransferases responsible for this modification represents a key step towards understanding its biological significance. Akr1p, one of the first identified protein palmitoyltransferases, is an 86 kDa yeast integral membrane protein. Using Akr1p purified from yeast, auto‐ and trans‐palmitoylation activity has been detected by the covalent modification of a substrate protein with a 3H‐labeled palmitoyl moiety in the presence of [3H]‐palmitoyl‐CoA and ATP. In addition to the previously identified substrate yeast casein kinase (Yck2), we have used mutagenesis to demonstrate that Akr1p catalyzes time‐dependent palmitoylation of two c‐terminal cysteines in the yeast protein, Ypl199c. We are examining the mechanism of Akr1p using mass spectrometry to identify covalent modifications and mutagenesis to examine the functional importance of the DHYC motif in a cysteine‐rich domain that is proposed as the active site of Akr1p.