The structure and function of pyruvate carboxylase

丙酮酸羧化酶的结构和功能

• 批准号: 7057881
• 负责人: WILLIAM Wallace CLELAND
• 金额: $ 27.98万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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/7057881
• 关键词: X ray crystallography; acetyl coA; active sites; adenosine triphosphate; binding sites; biotin; carboxylation; catalyst; crystallization; enzyme activity; enzyme mechanism; protein structure function; pyruvate carboxylase; site directed mutagenesis; three dimensional imaging /topography

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。