Molecular characterization, enzyme properties and transcriptional regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in a ruminal bacterium, Selenomonas ruminantium
N. Asanuma et T. Hino, Molecular characterization, enzyme properties and transcriptional regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in a ruminal bacterium, Selenomonas ruminantium, MICROBIO-UK, 147, 2001, pp. 681-690
elucidate the regulatory mechanism for propionate production in Selenomonas
ruminantium, the molecular properties and gene expression of phosphoenolpy
ruvate carboxykinase (Pck) and pyruvate kinase (Pyk) were investigated, The
Pck was deduced to consist of 538 aa with a molecular mass of 59.6 kDa, an
d appeared to exist as a monomer. The Pyk was revealed to consist of four i
dentical subunits consisting of 469 aa with a molecular mass of 51.3 kDa. B
oth Mg2+ and Mn2+ were required for the maximal activity of Pck, and Pck ut
ilized ADP, not CDP or IDP, as a substrate. Either Mg2+ or Mn2+ was require
d for Pyk activity, and the enzyme was activated by phosphoenolpyruvate (PE
P) and fructose 1,6-bisphosphate (FBP). Pyk activity was severely inhibited
by P-i, but restored by the addition of FBP. The K-m value of Pck for PEP
(0.55 mM) was nearly equal to the K-m value of Pyk for PEP, suggesting that
the partition of the flow from PEP in the fermentation pathways is determi
ned by the activity ratio of Pck to Pyk. Both pck and pyk genes were monoci
stronic, although two transcriptional start sites were found in pyk. The le
vel of pyk mRNA was not different whether glucose or lactate was the energy
substrate. However, the pck mRNA level was 12-fold higher when grown on la
ctate than on glucose. The level of pck mRNA was inversely related to the s
ufficiency of energy, suggesting that Pck synthesis is regulated at the tra
nscriptional level when energy supply is altered. It was conceivable that t
he transcription of pck in S. ruminantium is triggered by PEP and suppresse
d by ATP.