The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.
The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D013294:Lactococcus lactis / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000378:metabolism*
D002413:Cations, Divalent; D002414:Cations, Monovalent; D005636:Fructosephosphates; D003035:Cobalt; D008345:Manganese; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D000641:Ammonia; D003300:Copper; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium