Regulation of acetate kinase isozymes and its importance for mixed-acid fermentation in Lactococcus lactis.
Acetate kinase (ACK) converts acetyl phosphate to acetate along with the generation of ATP in the pathway for mixed-acid fermentation in Lactococcus lactis. The reverse reaction yields acetyl phosphate for assimilation purposes. Remarkably, L. lactis has two ACK isozymes, and the corresponding genes are present in an operon. We purified both enzymes (AckA1 and AckA2) from L. lactis MG1363 and determined their oligomeric state, specific activities, and allosteric regulation. Both proteins form homodimeric complexes, as shown by size exclusion chromatography and static light-scattering measurements. The turnover number of AckA1 is about an order of magnitude higher than that of AckA2 for the reaction in either direction. The Km values for acetyl phosphate, ATP, and ADP are similar for both enzymes. However, AckA2 has a higher affinity for acetate than does AckA1, suggesting an important role under acetate-limiting conditions despite the lower activity. Fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, and phospho-enol-pyruvate inhibit the activities of AckA1 and AckA2 to different extents. The allosteric regulation of AckA1 and AckA2 and the pool sizes of the glycolytic intermediates are consistent with a switch from homolactic to mixed-acid fermentation upon slowing of the growth rate.
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, and Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
Pranav
P
Puri
Anisha
A
Goel
Agnieszka
A
Bochynska
Bert
B
Poolman
D000084:Acetate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000143:Acids / Q000378:metabolism*; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005285:Fermentation; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D000143:Acids; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D000084:Acetate Kinase