The structure and function of pyruvate carboxylase

丙酮酸羧化酶的结构和功能

基本信息

批准号：
7418627
负责人：
WILLIAM Wallace CLELAND
金额：
$ 27.85万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-05-01 至 2009-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7418627
关键词：
Acetyl Coenzyme A Acetyl-CoA Carboxylase Active Sites Affect Affinity Labels Amino Acid Sequence Amino Acids Bicarbonates Binding Binding Sites Biotin Biotin carboxylase Carbamyl Phosphate Cessation of life Chemicals Chickens Chimera organism Coenzyme A Complex Condition Coupled Crystallization Data Depth Enzyme Kinetics Enzymes Escherichia coli Facility Construction Funding Category Family member Hepatic Holoenzymes Individual Infant Investigation Isoenzymes Isotopes Kinetics Label Ligase Liver Metabolism Methods MgADP Modification Movement Mutagenesis Mutate Mutation N1&apos -carboxybiotin Nature Non-Insulin-Dependent Diabetes Mellitus Numbers Obesity Oxaloacetates Physiological Play Positioning Attribute Prosthesis Protons Purpose Pyruvate Pyruvate Carboxylase Pyruvates Reaction Roentgen Rays Role Screening procedure Site Site-Directed Mutagenesis Solutions Source Structure Techniques Uncertainty Yeasts affinity labeling analog base carbonic acid, monoanhydride with phosphoric acid, ion(2-)carboxylate carboxylation enzyme mechanism inhibitor/antagonist insight mutant neonate oxaloacetate polypeptide reaction rate research study response thermophilic bacteria three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): Pyruvate carboxylase is a member of the family of biotin-dependent enzymes which play key roles in intermediary metabolism. Pyruvate carboxylase deficiencies mostly occur in neonates or infants and result in severe psychomotor retardation leading to early death. Over expression of hepatic pyruvate carboxylase is associated with type II diabetes and obesity. The purpose of this project is to determine the first three dimensional structure of pyruvate carboxylase as an example of an intact biotin-dependent holoenzyme, and to perform a detailed structure-function analysis on this enzyme. We shall: (1) Perform crystallization trials on pyruvate carboxylases from a number of sources with the aim of determining the 3-D structure of the enzyme by X-ray crystallographic analysis. (2) Determine the binding site of the physiological, allosteric activator, acetyl CoA by co-crystallization with a non-hydrolysable analogue and by affinity labeling. (3) Determine the regions of polypeptide sequence involved in the action of acetyl CoA by producing chimeric constructs of pyruvate carboxylases from species which show different degrees of response to acetyl CoA. By performing site-directed mutagenesis on specific amino acid residues in the enzyme and kinetic analyses of the wild-type and mutant enzymes using a combination of 2H, 13C and 18O kinetic isotope effect experiments, and steady-state and pre-steady state kinetic analysis to pinpoint the precise roles of these residues in the catalytic mechanism of the enzyme we shall elucidate (4) The catalytic mechanism of the biotin carboxylation reaction common to the majority of biotin-dependent carboxylases in the context of a holoenzyme. (5) The catalytic mechanism of the carboxylation of pyruvate by carboxybiotin and movement of carboxybiotin between the sites of the partial reactions in the enzyme active site. (6) The action of acetyl CoA at the level of the catalytic mechanism. (7) We shall synthesize carboxyphosphate and demonstrate its ability to act as an intermediate in the pyruvate carboxylase reaction.

描述（由申请人提供）：丙酮酸羧化酶是生物素依赖性酶家族的成员，其在中间代谢中发挥关键作用。丙酮酸羧化酶缺陷主要发生在新生儿或婴儿中，导致严重的精神运动迟缓，导致过早死亡。肝丙酮酸羧化酶的过度表达与 II 型糖尿病和肥胖有关。该项目的目的是确定丙酮酸羧化酶的第一个三维结构，作为完整的生物素依赖性全酶的例子，并对这种酶进行详细的结构功能分析。我们应： (1) 对多种来源的丙酮酸羧化酶进行结晶试验，目的是通过 X 射线晶体学分析确定酶的 3-D 结构。 (2) 通过与不可水解类似物共结晶并通过亲和标记来确定生理变构激活剂乙酰辅酶A的结合位点。 (3)通过从对乙酰辅酶A表现出不同程度的反应的物种产生丙酮酸羧化酶的嵌合构建体，确定参与乙酰辅酶A作用的多肽序列的区域。通过对酶中的特定氨基酸残基进行定点诱变，并结合2H、13C和18O动力学同位素效应实验以及稳态和稳态前动力学分析对野生型和突变型酶进行动力学分析为了查明这些残基在酶催化机制中的精确作用，我们将阐明 (4) 大多数生物素依赖性酶所共有的生物素羧化反应的催化机制全酶背景下的羧化酶。 (5)羧基生物素对丙酮酸羧化的催化机制以及羧基生物素在酶活性位点部分反应位点之间的移动。 (6)乙酰辅酶A在催化机制水平上的作用。 (7) 我们将合成羧磷酸盐并证明其作为丙酮酸羧化酶反应中间体的能力。