Pyruvate kinase isozymes from the green alga, Selenastrum minutum: II. Kinetic and regulatory properties

Abstract

The kinetic and regulatory properties of two pyruvate kinase isozymes, PK p and PK c (apparent chloroplastic and cytosolic isozymes, respectively) from the green alga Selenastrum minutum were studied. The two isozymes differed greatly in several kinetic properties. Although both isozymes showed hyperbolic substrate saturation kinetics, the apparent Michaelis constants for PEP and ADP were about twofold and fourfold lower, respectively, for PK c as compared with PK p. ADP was the preferred nucleotide substrate for both isozymes. However, PK c utilized alternate nucleotides far more effectively than did PK p. PK c and PK p also differed strongly in the effect of activators and inhibitors on the enzymes. Although both isozymes were activated by dihydroxyacetone phosphate (DHAP) with a similar activation constant of about 30 μ m, this activator (0.5 m m) caused an approximate 30% increase in the V max of PK c, but had no effect on the V max of PK p. PK p, but not PK c, was inhibited by ribose 5-phosphate, ribulose 1,5-bisphosphate, 2-phosphoglycerate, phosphoglycolate, and malate. Both isozymes were inhibited by MgATP, Mg 2citrate, Mg 2oxalate, and P i. PK c was far more sensitive to inhibition by P i, as compared with PK p. P i was a competitive inhibitor of PK c with respect to phospho enolpyruvate (PEP) ( K i = 1.3 mm). Glutamate was a potent inhibitor of PK c, but had no effect on PK p. In contrast with P i, glutamate was a mixed-type inhibitor of PK c with respect to PEP ( K i = 0.7 mm). DHAP facilitated the binding of PEP by both isozymes and reversed or relieved the inhibition of PK c by P i and/or glutamate. The regulatory properties of PK p indicate that it is likely less active in the light and more active in the dark. The in vivo activity of PK c is probably regulated by the relative cytosolic levels of DHAP, P i, and glutamate; this provides a rationale for the activation of algal cytosolic pyruvate kinase which occurs during periods of enhanced ammonia assimilation.