Mechanistic Studies of Protein Lipidation: Yeast Palmitoyltransferase Akr1pand Protein Farnesyltransferase.

Palmitoyltransferase;Akr1p;DHHC Motif;Auto-palmitoylation Site;Farnesyltransferase;Substrate Selectivity;Chemistry;Science;Chemistry

Guan, XiaomuRamamoorthy, Ayyalusamy ;

University of Michigan

关键词: Palmitoyltransferase;    Akr1p;    DHHC Motif;    Auto-palmitoylation Site;    Farnesyltransferase;    Substrate Selectivity;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/89718/xmguan_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Protein palmitoylation is a widespread lipid modification in which cysteine thiols on a substrate protein are modified with a palmitoyl group. Mutations in palmitoyltransferases responsible for this modification are associated with a number of neurological diseases and cancer progression. Defining the active site and catalytic mechanism of palmitoyltransferases 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. Mutagenesis studies of Akr1p suggest that a conserved DHYC motif serves as a potential active site; the hypothesized mechanism is a two-step mechanism where the palmitoyl group is transferred from palmitoyl-CoA to Akr1p, and finally from Akr1p to the substrate protein. A covalent intermediate has been detected using radioactive assays. In this study, we elucidated the role of each amino acid in the DHYC motif using mutagenesis. In addition, mutagenesis of all of the cysteine residues in Akr1p along with mass spectrometric analysis demonstrated that the DHYC cysteine is the site in Akr1p where a covalent thioester intermediate forms. Based on these data, we propose a detailed mechanism for palmitoylation catalyzed by Akr1p, which may shed light on the mechanism of other palmitoyltransferases within the DHHC protein family.Protein farnesylation is another important lipid modification in which the cysteine of a substrate protein is modified by attachment of a 15-carbon farnesyl group which results in membrane localization of the protein. Protein farnesyltransferase (FTase) catalyzes farnesylation of a specific C-terminal ;;Ca1a2X” sequence of substrate proteins. Here we analyze the determinants of recognition of the a2 residue by FTase, demonstrating that completely conserved tryptophan residues in FTase, although not essential for maintaining the farnesylation activity, play an important role in modulating the substrate selectivity of FTase. Mutagenesis studies demonstrate that the conserved W102β and W106β residues modulate both the reactivity of FTase and substrate selectivity based on the size of the binding pocket. The complete conservation of these two amino acids suggests that maintenance of the exact substrate selectivity of FTase is crucial for the in vivo activity.