The structure and function of pyruvate carboxylase

描述(申请人提供)：丙酮酸羧基酶是生物素依赖酶家族的成员，在中间代谢中起关键作用。丙酮酸羧酶缺乏症多见于新生儿或婴儿，可导致严重的精神运动迟缓，导致早逝。肝丙酮酸羧基酶的过度表达与II型糖尿病和肥胖有关。本项目的目的是确定丙酮酸羧化酶的第一个三维结构，作为一个完整的生物素依赖的全酶的例子，并对该酶进行详细的结构和功能分析。我们将：(1)对多种来源的丙酮酸羧基酶进行结晶实验，目的是通过X射线结晶学分析确定该酶的三维结构。(2)通过与非水解性类似物的共结晶和亲和标记，确定生理变构激活剂乙酰辅酶A的结合位置。(3)从对乙酰辅酶A反应程度不同的物种中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。通过对酶中特定的氨基酸残基进行定点突变，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，以确定这些残基在酶的催化机制中的确切作用，我们将阐明(4)在全酶的背景下，大多数生物素依赖的羧基酶共同的生物素羧化反应的催化机理。(5)羧基生物素对丙酮酸羧化反应的催化机理，以及羧基生物素在酶活性部位的部分反应部位之间的移动。(6)乙酰辅酶A在催化机理水平上的作用。(7)我们将合成羧基磷酸酯，并证明其在丙酮酸羧化反应中作为中间体的能力。

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.