STRUCTURE/REGULATION RELATIONSHIPS IN PEP CARBOXYLASE

PEP 羧化酶的结构/调控关系

• 批准号: 6481216
• 负责人: SCOTT D GROVER
• 金额: $ 7.33万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481216
• 关键词: Escherichia coli; X ray crystallography; affinity labeling; allosteric site; chemical association; chemical structure function; corn; enzyme activity; enzyme inhibitors; enzyme structure; glucose 6 phosphate; ionizing radiation; phosphoenolpyruvate carboxylase; phosphorylation; polymerase chain reaction; protein sequence; site directed mutagenesis; stoichiometry

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.

玉米磷酸烯醇式丙酮酸（PEP）羧化酶的研究将进行映射的残基参与结合抑制剂苹果酸，并探测变构效应和调节磷酸化之间的相互作用的物理基础。将寻求制备磷酸化和脱磷酸化形式的酶的衍射质量晶体的条件。与细菌形式的酶不同，其影响Ser 15的磷酸化和几种变构效应物。磷酸化不是必需的，因为酶的形式对苹果酸的抑制不太敏感，而对激活剂葡萄糖-6磷酸更敏感。基于酵母糖原磷酸化酶中变构调节和可逆磷酸化之间相互作用的物理基础的最新发现（Lin et al. 1996），以及最近发表的磷酸化丝氨酸和变构抑制位点处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酶的敏感性蛋白磷酸酶去磷酸化。我们将通过使用33 P标记的重组玉米PEP羧化酶和2A型蛋白磷酸酶在体外监测磷酸酶活性来测试该模型。在存在和不存在变构抑制剂和活化剂的情况下，以及在已知调节变构行为的pH范围内，从磷酸化酶释放33 PO 4。定点诱变将用于探测酶区域中似乎参与抑制剂结合的各种残基的功能作用。这些结果有望为了解酶的共价和非共价调节机制的相互作用提供一般性的见解，并具体介绍这种酶的抑制剂反应，尽管它在高等动物中没有发现，但在细菌，植物和寄生生物中起着关键作用。