K. Winzer et al., ACETATE KINASE FROM CLOSTRIDIUM-ACETOBUTYLICUM - A HIGHLY SPECIFIC ENZYME THAT IS ACTIVELY TRANSCRIBED DURING ACIDOGENESIS AND SOLVENTOGENESIS, Microbiology, 143, 1997, pp. 3279-3286
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.