STRUCTURE/REGULATION RELATIONSHIPS IN PEP CARBOXYLASE

研究人员将对玉米磷酸烯醇丙酮酸（PEP）羧化酶进行研究，以绘制与苹果酸抑制剂结合的残基，并探索变构效应物与调控磷酸化之间相互作用的物理基础。将寻求制备酶的磷和去磷形式的衍射质量晶体的条件。不像细菌形式的酶，它的作用是磷酸化在Ser15和几个变构效应。不需要磷酸化，因为酶的形式对苹果酸盐的抑制不太敏感，而对激活剂葡萄糖-6磷酸更敏感。基于最近对酵母糖原磷酸化酶变构调节和可逆磷酸化之间相互作用的物理基础的发现（Lin et al. 1996），以及最近发表的磷酸化丝氨酸与变构抑制位点抑制剂之间的竞争性相互作用。该模型预测PEP羧化酶的变构效应调节了该酶对蛋白磷酸酶去磷酸化的敏感性。我们将使用33p标记的重组玉米PEP羧化酶和2A型蛋白磷酸酶，通过体外监测磷酸酶活性来测试该模型。33PO4从磷酸化酶的释放将在存在或不存在变抗抑制剂和活化剂以及已知调节变抗行为的pH范围内进行。位点定向诱变将用于探测酶的各种残基的功能作用，这些残基似乎参与了抑制剂的结合。该研究结果有望为进一步了解酶的共价和非共价调控机制提供新的视角，并对该酶的抑制剂反应进行具体介绍，该酶虽未在高等动物中发现，但在细菌、植物和寄生生物中起着关键作用。

Studies of the enzyme phosphoenolpyruvate (PEP) carboxylase from maize will be conducted to map the residues involved in the binding the inhibitor malate, and to probe the physical basis of the interaction between allosteric effectors and regulatory phosphorylation. Conditions for preparing diffraction-quality crystals of the phospho and dephospho forms of the enzymes will be sought. Unlike the bacterial form of the enzyme, which effects of phosphorylation at Ser15 and several allosteric effectors. Phosphorylation is not required for form of the enzyme is less sensitive to inhibition by malate and more sensitive to the activator glucose-6 phosphate. Based on recent findings on the physical basis for interaction between allosteric regulation and reversible phosphorylation in yeast glycogen phosphorylase (Lin et al. 1996), and the recently published competitive interaction between the phosphorylated serine and the inhibitor at the allosteric inhibition site. The model predicts that the allosteric effectors of PEP carboxylase modulate the enzyme's susceptibility to dephosphorylation by protein phosphatase. We will test this model by monitoring phosphatase activity in vitro using 33P-labeled recombinant maize PEP carboxylase and type 2A protein phosphatase. The release of 33PO4 from the phosphorylated enzyme will be followed in the presence and absence of allosteric inhibitors and activators and over a range of pH known to modulate allosteric behavior. Site-directed mutagenesis will be used to probe the functional role of various residues in regions of the enzyme that appear to be involved in inhibitor binding. The results are expected to provide insight into the interplay of covalent and non-covalent regulatory mechanisms of enzymes in general, and specific introduction about the inhibitor response of this enzyme which, though it is not found in higher animals, plays a key role in bacteria, plants and parasitic organisms.