Lin M, Turpin D H, Plaxton W C
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Arch Biochem Biophys. 1989 Feb 15;269(1):228-38. doi: 10.1016/0003-9861(89)90104-5.
The kinetic and regulatory properties of two pyruvate kinase isozymes, PKp and PKc (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 PKc as compared with PKp. ADP was the preferred nucleotide substrate for both isozymes. However, PKc utilized alternate nucleotides far more effectively than did PKp. PKc and PKp 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 microM, this activator (0.5 mM) caused an approximate 30% increase in the Vmax of PKc, but had no effect on the Vmax of PKp. PKp, but not PKc, was inhibited by ribose 5-phosphate, ribulose 1,5-bisphosphate, 2-phosphoglycerate, phosphoglycolate, and malate. Both isozymes were inhibited by MgATP, Mg2citrate, Mg2oxalate, and Pi. PKc was far more sensitive to inhibition by Pi, as compared with PKp. Pi was a competitive inhibitor of PKc with respect to phosphoenolpyruvate (PEP) (Ki = 1.3 mM). Glutamate was a potent inhibitor of PKc, but had no effect on PKp. In contrast with Pi, glutamate was a mixed-type inhibitor of PKc with respect to PEP (Ki = 0.7 mM). DHAP facilitated the binding of PEP by both isozymes and reversed or relieved the inhibition of PKc by Pi and/or glutamate. The regulatory properties of PKp indicate that it is likely less active in the light and more active in the dark. The in vivo activity of PKc is probably regulated by the relative cytosolic levels of DHAP, Pi, and glutamate; this provides a rationale for the activation of algal cytosolic pyruvate kinase which occurs during periods of enhanced ammonia assimilation.
对来自绿藻微小色球藻的两种丙酮酸激酶同工酶PKp和PKc（分别为表观叶绿体和胞质同工酶）的动力学和调节特性进行了研究。这两种同工酶在几个动力学特性上有很大差异。尽管两种同工酶都表现出双曲线型底物饱和动力学，但与PKp相比，PKc对磷酸烯醇式丙酮酸（PEP）和ADP的表观米氏常数分别低约两倍和四倍。ADP是两种同工酶的首选核苷酸底物。然而，PKc利用替代核苷酸的效率比PKp高得多。PKc和PKp在激活剂和抑制剂对酶的作用方面也有很大差异。尽管两种同工酶都被磷酸二羟丙酮（DHAP）激活，其激活常数相似，约为30微摩尔，但这种激活剂（0.5毫摩尔）使PKc的Vmax增加了约30%，但对PKp的Vmax没有影响。PKp被5-磷酸核糖、1,5-二磷酸核酮糖、2-磷酸甘油酸、磷酸乙醇酸和苹果酸抑制，而PKc不受影响。两种同工酶都被MgATP、Mg2柠檬酸盐、Mg2草酸盐和Pi抑制。与PKp相比，PKc对Pi抑制的敏感性要高得多。Pi是PKc对磷酸烯醇式丙酮酸（PEP）的竞争性抑制剂（Ki = 1.3毫摩尔）。谷氨酸是PKc的有效抑制剂，但对PKp没有影响。与Pi不同，谷氨酸是PKc对PEP的混合型抑制剂（Ki = 0.7毫摩尔）。DHAP促进了两种同工酶对PEP的结合，并逆转或减轻了Pi和/或谷氨酸对PKc的抑制。PKp的调节特性表明，它在光照下可能活性较低，在黑暗中活性较高。PKc在体内的活性可能受胞质中DHAP、Pi和谷氨酸相对水平的调节；这为藻类胞质丙酮酸激酶在氨同化增强期间的激活提供了一个理论依据。
