Kinetic and molecular characteristics of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L

Abstract

1. 1.At low phosphopyruvate concentrations, fructose 1,6-diphosphate enhances the activity of pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from the adductor muscle of sea mussels. At pH 7.6 [phosphopyruvate] - 0.2 mM and [ADP] = 5 mM, half-maximal stimulation is reached with [ Fru-1,6-P 2] = 2 μM; at ph 6.7 it is reached with [ Fru-1,6-P 2] = 15 μM 2. 2.Glucose 1,6-diphosphate enhances enzyme activity but less so than Fru-1,6-P 2 . Cyclic AMP stimulates to a even lesser extent 3. 3.The reaction rate is inhibited when the concentration of the co-substrate ADP is substantially higher than phosphopyruvate. The Lineweaver-Burk plots for ADP at fixed concentrations of phosphopyruvate show no cooperative effects, whilst similar plots for phosphopyruvate at fixed concentrations of ADP all show positive cooperativity 4. 4.Alanine strongly inhibits enzyme activity. At pH 6.7 [phosphopyruvate] = 0.2 mM and [ADP] - 2 mM, 50% inhibition is reached with [alanine] = 0.1 mM; at pH 7.6 it is reached at [alanine] - 1 mM. 0.1 mM Fru-1,6-P 2 counteracts this inhibition giving a hyperbolic substrate-saturation curve 5. 5.ATP inhibition is strongly pH-dependant. At pH 6.7, [phosphopyruvate] = 0.2. mM and [ADP] = 2 mM, 50% inhibition is reached at [ATP] = 0.62 mM. At pH 7.6 there is a little influence of ATP. Fru-1,6-P 2 also cancels this inhibition. Other trinucleotides and AMP inhibit to a less extent 6. 6.The molecular mass of the enzyme was found to be 220 000 ± 10%. On electrofocusing, one main peak is found with pI 6.70. Three smaller peaks have pI values of 6.44, 6.59 and 6.83.