Biophysical Studies of Human Malic Enzymes

苹果酸酶(ME)催化苹果酸氧化脱羧生成丙酮酸和二氧化碳，伴随着辅因子NAD或NADP的还原。它们通常是同系四聚体，单体的相对分子质量约为60kD。到目前为止，在哺乳动物中已鉴定出三种苹果酸酶同工酶--胞浆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在三种异构体中有几个独特的特征。它是一种合作酶，相对于底物苹果酸表现出S型动力学。其活性受变构控制，富马酸为激活剂，三磷酸腺苷为抑制剂。它可以同时使用NAD和NADP作为辅助因子，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。作为更好地了解这些酶的第一步，最近测定了人m-NAD-ME与NAD的络合物的晶体结构。结果表明，苹果酸酶是一类新的氧化脱羧酶。这第一种结构代表了进一步的生化、结构和生物物理研究的起点，以表征这一重要类别的酶。将特别强调确定苹果酸酶的一般催化机制，特别是描述m-NAD-ME独特变构性质的结构基础。研究的另一个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。还将获得依赖于NADP的异构体的结构，以提供更多信息进行比较。结构分析的结果将通过诱变和动力学研究进行评估。苹果酸酶广泛分布于自然界，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。拟议的研究应有助于更好地了解这类重要酶的生化特性和生物学功能。这项研究还应提供关于变构控制酶活性的结构基础以及脱氢酶和脱羧酶的一般催化机制的更多见解。

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.