2015
Taylor, Martin S; Dempsey, Daniel R; Hwang, Yousang; Chen, Zan; Chu, Nam; Boeke, Jef D; Cole, Philip A
Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs Journal Article
In: Bioorg Chem, vol. 62, pp. 64-73, 2015, ISSN: 1090-2120.
Abstract | Links | BibTeX | Tags: Acyltransferases, Amino Acid Sequence, Animals, Baculoviridae, Biotin, Cell Line, Enzyme Assays, Genetic Vectors, Ghrelin, Hydrogen-Ion Concentration, Mice, Models, Molecular
@article{1299970,
title = {Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs},
author = {Martin S Taylor and Daniel R Dempsey and Yousang Hwang and Zan Chen and Nam Chu and Jef D Boeke and Philip A Cole},
doi = {10.1016/j.bioorg.2015.07.003},
issn = {1090-2120},
year = {2015},
date = {2015-10-01},
journal = {Bioorg Chem},
volume = {62},
pages = {64-73},
abstract = {Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.},
keywords = {Acyltransferases, Amino Acid Sequence, Animals, Baculoviridae, Biotin, Cell Line, Enzyme Assays, Genetic Vectors, Ghrelin, Hydrogen-Ion Concentration, Mice, Models, Molecular},
pubstate = {published},
tppubtype = {article}
}
Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0-9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.