3/20/2024 0 Comments Kaleidagraph software enzymes![]() ![]() An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP i. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP + to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase 24. In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann 17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD + by the enzymes pyruvate kinase and lactate dehydrogenase 21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine 23. An acetate kinase which can only utilize PP i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus 15,16. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila 7-14. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya 6. Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily 1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. ![]()
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