The absorption coefficient of butyryl-CoA dehydrogenase from Megasphaera elsdenii at 450nm is determined as 14.4mM-1 cm-1 in the CoA-free form and 14.2mM-1 cm-1 in the CoA-liganded form (both yellow). The latter value is considerably higher than the earlier published estimate. Phenazine ethosulphate offers great advantages over phenazine methosulphate as a coupling dye in the catalytic assay de...

Peptides and proteins fragment sequence-specifically in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions.

Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics stu...

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-contain...

We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used th...

Herein, we describe a practical, one-pot variant of the sulfo-click reaction, in which 9-fluorenylmethyl-protected thioesters are rapidly deprotected and reacted further with sulfonylazides to give N-acyl sulfonamides.

The fatty acid:CoA ligase (acyl-CoA synthetase, EC 6.2.1.3) of rat liver microsomes was solubilized with Triton X-100 and bound to Matrex Gel Red A. Fatty acid:CoA ligase immobilized on Matrex Gel Red A was active and proved useful for the synthesis of fatty acyl-CoA thioesters. The immobilized activity was characterized by a 3-fold higher apparent Km for ATP than the soluble activity, similar ...