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Catalysis of ester hydrolysis by Escherichia coli carboxyltransferase
Author(s) -
Thalji Nabil,
Waldrop Grover L
Publication year - 2009
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.23.1_supplement.502.13
Subject(s) - chemistry , hydrolysis , pyruvate carboxylase , acetyl coa carboxylase , biotin , oxyanion hole , substrate (aquarium) , acetyl coa , biochemistry , catalysis , stereochemistry , enzyme , active site , biology , ecology
Acetyl‐CoA carboxylase (ACCase) catalyzes the biotin‐dependent carboxylation of acetyl‐CoA to malonyl‐CoA in the first step of fatty acid synthesis in all organisms. Bacterial ACCase consists of two catalytic components: biotin carboxylase (BC), which catalyzes the carboxylation of biotin, and carboxyltransferase (CT) which transfers CO 2 from carboxybiotin to acetyl‐CoA to make malonyl‐CoA. The binding sites in CT for carboxybiotin and acetyl‐CoA possess oxyanion holes that are thought to stabilize oxyanionic intermediates during the transfer. We investigated whether these oxyanion holes catalyze ester hydrolysis, which also proceeds through an oxyanionic transition state, using p ‐nitrophenyl acetate (PNPA) as an alternative substrate for E. coli CT. By measuring production of p ‐nitrophenol (PNP) spectrophotometrically it was found that CT catalyzes PNPA hydrolysis at pH 7.0. Biotin inhibited hydrolysis of PNPA, while malonyl‐CoA increased the rate of hydrolysis. Mutation of an oxyanion hole residue in the acetyl‐CoA binding site did not affect the rate of catalysis. Thus, PNPA hydrolysis likely occurs in the biotin binding site, and binding of acetyl‐CoA enhances binding of PNPA to the enzyme.