Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of the enzymic reaction responsible for chloramphenicol resistance in bacteria was examined by steady-state kinetic methods. The forward reaction catalysed by chloramphenicol acetyltransferase leads to inactivation of the antibiotic. Use of alternative acyl donors and acceptors, as well as the natural substrates, has yielded data that favour the view that the reaction proceeds to the formation of a ternary complex by a rapid-equilibrium mechanism wherein the addition of substrates may be random but a preference for acetyl-CoA as the leading substrate can be detected. Chloramphenicol and acetyl-CoA bind independently, but the correlation between directly determined and kinetically derived dissociation constants is imperfect because of an unreliable slope term in the rate equation. The reverse reaction, yielding acetyl-CoA and chloramphenicol, was studied in a coupled assay involving citrate synthase and malate dehydrogenase, and is best described by a rapid-equilibrium mechanism with random addition of substrates. The directly determined dissociation constant for CoA is in agreement with that derived from kinetic measurements under the assumption of an independent-sites model.
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PMID:Analysis of the mechanism of chloramphenicol acetyltransferase by steady-state kinetics. Evidence for a ternary-complex mechanism. 659 36

ATP-citrate lyase (CL) catalyzes the conversion of citrate and CoA to oxaloacetate (OA) and acetyl-CoA. As the coupled malic dehydrogenase (MDH) assay is not able either to study the effect of oxaloacetate (OA) on CL activity or to measure accurately CL activity in biological samples, a new assay was developed. The CL-citrate coupled CAT assay measures the amount of acetyl-CoA formed by transferring radiolabeled acetyl-CoA synthesized from [14C]citrate to chloramphenicol with chloramphenicol acetyltransferase (CAT). Employing this assay, the rate of increase in acetyl-CoA synthesis from citrate is linear with respect to added CL. Kinetic values for ATP, CoA and citrate are similar to those obtained using the MDH assay. The effect of CL phosphorylation on enzyme activity was determined. CL phosphorylated by cAMP-dependent protein kinase or by this kinase and glycogen synthase kinase-3 (GSK-3) decreases the apparent Vmax without changing the apparent Km. The effect of OA, a product of the enzyme reaction, on CL activity was also determined. Computational analysis of the data obtained without added OA and at three concentrations of OA indicate that the apparent Km for the substrate is not altered even though the apparent Vmax is decreased. The effect of OA on the activity of phosphorylated enzyme was also determined. OA decreases the apparent Vmax of the phosphorylated enzyme to the same extent as in control CL. This assay is able to measure CL activity in cytosol from 3T3-L1 adipocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of oxaloacetate and phosphorylation on ATP-citrate lyase activity. 766 53