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Mechanistic Enzyme Studies of Acyl‐CoA Synthetase
Author(s) -
Melton Elaina Marie,
Li Hong,
Quackenbush Steven,
DiRusso Concetta,
Black Paul N
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a953-a
Subject(s) - acylation , enzyme , chemistry , biochemistry , fatty acid , kinetics , yeast , size exclusion chromatography , dimer , substrate (aquarium) , enzyme kinetics , enzyme activator , stereochemistry , catalysis , active site , biology , organic chemistry , ecology , physics , quantum mechanics
Fatty Acyl‐CoA Synthetase (ACSL) is a central component in the process of vectorial acylation, which links fatty acid import with activation. The mechanism by which ACSL activates fatty acids (FA) to acyl‐CoAs for metabolic utilization has not been completely defined. Studies from our laboratory have shown the yeast fatty acid transport protein functions in concert with a cognate ACSL to facilitate vectorial acylation. In order to understand the role of ACSL in this process we have defined the mechanism and the kinetic parameters, which govern the reaction. The yeast ASCL Faa1p was expressed in BL21(DE3) and purified to homogeneity using nickel affinity and gel filtration chromatography. The purified Faa1p is a dimer and is homogeneous using analytical ultracentrifugation. The enzymatic properties of Faa1p were examined with respect to pH and temperature optima and metal ion requirements. Faa1p had a maximal activity using oleate as substrate at a pH of 8.0. The enzyme was stable at temperatures between 20°C and 42°C, with maximal activity at 30°C. Faa1p has specificity for long chain FA when compared to medium and very long chain FA. The catalytic mechanism is currently being investigated by steady‐state kinetics. Initial velocity studies and product‐inhibition pattern with AMP and PPi will give insights into the mechanism of long‐chain FA activation. Supported by NIH grant RO1‐GM056840.