Biophysical Studies of Human Malic Enzymes

TongMCB 9974700苹果酸酶（ME）催化苹果酸氧化脱羧产生丙酮酸和CO2，同时伴随辅因子NAD或NADP的还原。它们通常是均四聚体，单体的分子量约为60 kD。在哺乳动物中，迄今为止已经鉴定出苹果酸酶的三种同种型-细胞溶质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在三种同种型中具有几个独特的特征。它是一种协同酶，对底物苹果酸表现出S形动力学。其活性受别构控制，富马酸盐作为激活剂，ATP作为抑制剂。它可以使用NAD和NADP作为辅因子，尽管在生理条件下它更喜欢NAD。这些独特性质的结构基础目前尚不清楚。作为对这些酶更深入了解的第一步，最近确定了与NAD复合的人m-NAD-ME的晶体结构。结构表明苹果酸酶是一类新的氧化脱羧酶。这第一个结构代表了进一步的生化，结构和生物物理研究的起点，以表征这类重要的酶。特别强调将被放置在定义一般的苹果酸酶的催化机制，特别是在描绘的结构基础上的独特的变构特性的m-NAD-ME。研究的另一方面将集中在苹果酸酶的辅因子选择性的结构基础上。m-NAD-ME为研究NAD和NADP与同一酶的结合提供了独特的机会。还将获得NADP依赖性亚型的结构，以提供用于比较的额外信息。 苹果酸酶广泛存在于自然界中，在细菌、酵母、真菌、植物和动物中均有发现。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。拟议中的研究将导致对这类重要酶的生化特性和生物学功能的更深入了解。这项研究还应该提供更多的见解的结构基础上的变构控制酶的活动和催化机制的脱氢酶和脱羧酶一般。

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.