[13] Acetyl-CoA Synthetases I and II from Pyrococcus furiosus

Publisher Summary Acetate and acetyl-CoA are important intermediates in microbial metabolism. Fatty acids, polysaccharides, and proteins are all broken down into acetyl-CoA units, a requisite step prior to energy generation. Acetyl CoA is used as a building block in the synthesis of various cell components or it is transformed to acetate as an end-product of certain fermentative pathways. The conversion of acetyl-CoA to acetate is, therefore, a key step in general metabolism. In most bacteria, this transformation is catalyzed by two enzymes: phosphoacetyltransferase and acetate kinase. Two distinct enzymes with ADP-dependent acetyl-CoA synthetase activity have been purified from cell-free extracts of P. furiosus , an organism that grows by fermenting both sugars and peptides. These enzymes convert the acetyl-CoA produced by the fermentative pathways into acetate with the concomitant production of ATP. Acetyl-CoA is produced by the oxidative decarboxylation of pyruvate by pyruvate ferredoxin oxidoreductase (POR). However, the two enzymes, which are termed acetyl-CoA synthetase (ACS) I and II, differ in their substrate specificity. This chapter describes the methods used to assay and purify ACS I and ACS II from P. furiosus , along with some of their molecular and catalytic properties.