ACETATE KINASE FROM CLOSTRIDIUM-ACETOBUTYLICUM - A HIGHLY SPECIFIC ENZYME THAT IS ACTIVELY TRANSCRIBED DURING ACIDOGENESIS AND SOLVENTOGENESIS

Acetate kinase (ATP: phosphotransferase, EC 2.7.2.1) has been purified 294-fold from acid-producing cells of Clostridium acetobutylicum DSM 1731 to a specific activity of 1087 U mg(-1) (ADP-forming direction). The dimeric enzyme consisted of subunits with a molecular mass of 43 k Da. The molecular mass of the native acetate kinase was in the range 8 7-94 kDa as judged by gel filtration and native gel electrophoresis. T he enzyme showed high specificity for the substrates acetate and ATP, and maximal activity was obtained with Mn2+ as divalent cation. The pr esence of mercury compounds such as HgCl2 and p-hydroxymercuribenzoate resulted in an essential loss of activity. The apparent K-m values fo r acetate, Mg-ATP, acetyl phosphate, and Mg-ADP were 73, 0.37, 0.58 an d 0.71 mM. An activity-staining procedure for detection of acetate kin ase in crude cell extracts after separation on native polyacrylamide g els was developed. A DNA fragment encoding 246 bp of the acetate kinas e gene of C. acetobutylicum DSM 792 was cloned by a PCR-based approach . Northern blot analysis revealed transcription of the gene under acid -and solvent-producing conditions with no significant differences at t he level of transcription.