Roeske C A, Chollet R
J Biol Chem. 1987 Sep 15;262(26):12575-82.
The active site(s) of the bifunctional regulatory protein of pyruvate,orthophosphate dikinase catalyze(s) the Pi-dependent activation (dephosphorylation) and ADP-dependent inactivation (phosphorylation) of maize leaf dikinase. The chemical modification studies of the regulatory protein active sites presented in this paper are interpreted as showing the two sites to be physically distinct. Pyridoxal 5'-phosphate and 2-nitro-5-thiocyanatobenzoate (NTCB) selectively inhibit the dikinase activating site, which is protected by the nonprotein substrate, Pi. Phenylglyoxal blocks both the activation and inactivation sites; the former is protected selectively by Pi and the latter by both the nonprotein substrate, ADP, and Pi. The Pi that protects the inactivation site is distinct from the activation substrate. Inhibition studies show Pi to be a parabolic competitive inhibitor of the ADP-dependent inactivation of dikinase, implying that besides substrate Pi, a second phosphate also binds to the regulatory protein. The above chemical modifications are not mutually exclusive; neither NTCB, 5,5'-dithiobis-(2-nitrobenzoate), nor pyridoxal 5'-phosphate blocks subsequent modification of the activation site by phenylglyoxal. Similarly, prior modification with NTCB does not affect modification by pyridoxal 5'-phosphate.
丙酮酸、正磷酸二激酶双功能调节蛋白的活性位点催化玉米叶片二激酶的磷酸根依赖性激活（去磷酸化）和ADP依赖性失活（磷酸化）。本文中对调节蛋白活性位点的化学修饰研究表明这两个位点在物理上是不同的。磷酸吡哆醛和2-硝基-5-硫氰酸苯甲酸（NTCB）选择性抑制二激酶激活位点，该位点受到非蛋白质底物磷酸根的保护。苯乙二醛阻断激活和失活位点；前者受到磷酸根的选择性保护，后者受到非蛋白质底物ADP和磷酸根的保护。保护失活位点的磷酸根与激活底物不同。抑制研究表明，磷酸根是二激酶ADP依赖性失活的抛物线型竞争性抑制剂，这意味着除了底物磷酸根外，第二个磷酸根也与调节蛋白结合。上述化学修饰并非相互排斥；NTCB、5,5'-二硫代双（2-硝基苯甲酸）或磷酸吡哆醛均不阻断随后苯乙二醛对激活位点的修饰。同样，先用NTCB修饰并不影响磷酸吡哆醛的修饰。
