Biophysical Studies of Human Malic Enzymes

人类苹果酸酶的生物物理学研究

• 批准号: 9974700
• 负责人: Liang Tong
• 金额: $ 50万
• 依托单位: Columbia University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974700&HistoricalAwards=false
• 关键词: Biophysical; Studies; Human; Malic; Enzymes

TongMCB9974700Malic enzymes (ME) catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2, with the concomitant reduction of the cofactor NAD or NADP. They are usually homo-tetramers, with molecular weights of about 60kD for the monomer. In mammals, three isoforms of malic enzymes have been identified so far - cytosolic NADP-dependent ME (c-NADP-ME), mitochondrial NADP-dependent ME (m-NADP-ME), and mitochondrial NAD (P)- dependent ME (m-NAD-ME). m-NAD-ME has several unique features among the three isoforms. It is a cooperative enzyme and exhibits sigmoidal kinetics with respect to the substrate malate. Its activity is allosterically controlled, with fumarate as an activator and ATP as an inhibitor. It can use both NAD and NADP as the co-factor, although it prefers NAD under physiological conditions. The structural basis for these unique properties are currently unknown. As a first step towards a greater understanding of these enzymes, the crystal structure of human m-NAD-ME in complex with NAD was determined recently. The structure demonstrates that malic enzymes are a new class of oxidative decarboxylases. This first structure represents the starting point for further biochemical, structural, and biophysical studies to characterize this important class of enzymes. Special emphasis will be placed on defining the catalytic mechanisms of malic enzymes in general, and on delineating the structural basis for the unique allosteric properties of m-NAD-ME in particular. Another aspect of the research will focus on the structural basis for the co-factor selectivity of the malic enzymes. m-NAD-ME offers the unique opportunity of studying the binding of NAD and NADP to the same enzyme. Structures of the NADP-dependent isoforms will also be obtained to provide additional information for comparison. The results obtained from the structural analyses will be assessed by mutagenesis and kinetic studies.Malic enzymes are widely distributed in nature, having been found in bacteria, yeast, fungi, plants, and animals. Their amino acid sequences are highly conserved; suggesting that malic enzymes may have important biological functions. The proposed research should lead to a greater understanding of the biochemical properties and biological functions of this important class of enzymes. The research should also provide additional insights on the structural basis for the allosteric control of enzyme activities and the catalytic mechanisms of dehydrogenases and decarboxylases in general.

TongMCB9974700苹果酸酶 (ME) 催化苹果酸氧化脱羧产生丙酮酸和 CO2，同时还原辅因子 NAD 或 NADP。它们通常是同源四聚体，单体分子量约为 60kD。在哺乳动物中，迄今为止已鉴定出三种苹果酸酶亚型：胞质 NADP 依赖性 ME (c-NADP-ME)、线粒体 NADP 依赖性 ME (m-NADP-ME) 和线粒体 NAD (P) 依赖性 ME (m-NAD-ME)。 m-NAD-ME 在三种异构体中具有几个独特的特征。它是一种协同酶，相对于底物苹果酸表现出 S 形动力学。其活性受到变构控制，富马酸盐作为激活剂，ATP 作为抑制剂。它可以同时使用 NAD 和 NADP 作为辅因子，尽管它在生理条件下更喜欢 NAD。这些独特性质的结构基础目前尚不清楚。作为进一步了解这些酶的第一步，最近确定了人 m-NAD-ME 与 NAD 复合物的晶体结构。该结构表明苹果酸酶是一类新型氧化脱羧酶。第一个结构代表了进一步生化、结构和生物物理研究的起点，以表征这一类重要的酶。将特别强调定义苹果酸酶的催化​​机制，特别是描述 m-NAD-ME 独特变构特性的结构基础。研究的另一个方面将集中于苹果酸酶的辅因子选择性的结构基础。 m-NAD-ME 提供了研究 NAD 和 NADP 与同一酶结合的独特机会。还将获得 NADP 依赖性亚型的结构，以提供用于比较的附加信息。 结构分析获得的结果将通过诱变和动力学研究进行评估。苹果酸酶在自然界中广泛分布，已在细菌、酵母、真菌、植物和动物中发现。它们的氨基酸序列高度保守；表明苹果酸酶可能具有重要的生物学功能。拟议的研究应该有助于更好地了解这一类重要酶的生化特性和生物功能。该研究还应为酶活性变构控制的结构基础以及脱氢酶和脱羧酶的一般催化机制提供更多见解。