Activation of acetate in acetate-grown Methanosarcina thermophila: purificationm and characterization of acetate kinase

Extracts of acetate-grown <u>Methanosarcina thermoghila</u> were assayed for the presence of enzymes which might catalyze a proposed activation of acetate as the initial step in the pathway of methanogenesis from acetate by that organism. Acetate kinase and phosphate acetyltransferase activities of 4.9 and 49 μmoles of product/min/mg protein, respectively, were detected. Acetate kinase was purified 102- fold to a specific activity of 656 μmoles ADP formed/min/mg protein and was essentially homogeneous by denaturing gel electrophoresis. The native enzyme (Mr 94,000) was an α₂ homodimer with a subunit Mr of 53,000. Activity was optimal between pH 7.0 and 7.4 and was stable to heating at 70°C for 15 min. The apparent Km for acetate was 22 mM (Vmax = 668 μmoles ADP/min/mg protein) and 2.8 mM for ATP (Vmax = 777 pmoles ADP/min/ protein). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. One of several divalent cations was required for activity; the maximum rate was obtained with Mn2+.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Anaerobic Microbiology

Department dc:contributor.department

Anaerobic Microbiology

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

1980

Author dc:creator

• Aceti, David John

Chair dc:contributor.committeechair

• Ferry, James G.

Committee members dc:contributor.committeemember

• Wilkins, Tracy D.
• Dean, Dennis R.
• White, Robert H.

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

etd-02072013-040125

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/40983