Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism.
Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism.
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DOI：
10.1021/bi1012518
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发表时间：
2010-12-14
期刊：
影响因子：
2.9
通讯作者：
Sauve, Anthony A.
中科院分区：
文献类型：
--
作者：
French, Jarrod B.;Cen, Yana;Vrablik, Tracy L.;Xu, Ping;Allen, Eleanor;Hanna-Rose, Wendy;Sauve, Anthony A.
烟酰胺酶是将烟酰胺水解为烟酸的代谢酶。这些酶广泛分布在生物体中，例如在分枝杆菌、真细菌、原生动物、酵母和无脊椎动物的基因组中发现编码，但在哺乳动物中没有发现。虽然最近的结构工作提高了对这些酶的理解，但它们的催化机制仍然没有很好地理解。最近的数据表明，烟酰胺酶是几种病原微生物的生长和毒力所必需的。酿酒酵母、黑腹果蝇和秀丽隐杆线虫的酶调节各自生物体的寿命，这与NAD+代谢和生物体衰老调节中的作用一致。本文测定了C. elegans，S.报道了酿酒酵母、肺炎链球菌（Streptococcus pneumoniae）（引起人类肺炎的病原体）、伯氏疏螺旋体（Borrelia burgdorferi）（引起莱姆病的病原体）和恶性疟原虫（Plasmodium falciparum）（引起大多数人类疟疾）。烟酰胺酶通常是有效的催化剂，稳态kcat值通常超过1 s-1。烟酰胺的Km值较低，范围为2 - 110 µM。烟碱醛被确定为这些酶的有效竞争性抑制剂，在低µM至低nM范围内结合所有检测的烟酰胺酶。合成了多种烟醛衍生物，并在动力学测定中作为抑制剂进行了评价。抑制作用与每种酶上的普遍保守的催化Cys与醛羰基碳反应形成硫代半缩醛复合物是一致的，该复合物由保守的含氧阴离子空穴稳定。色葡萄pneumoniae烟酰胺酶可以催化18 O-水将18 O交换成烟酸的羧基氧。收集的数据，沿着几个突变体的动力学分析，使我们能够提出一个催化机制，解释烟酰胺酶和烟酸18 O交换化学的S。肺炎球菌酶的一个重要组成部分，涉及关键催化残基、催化过渡金属离子和硫酯中间体的中间性。
Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast and invertebrates but there are none found in mammals. Although recent structural work has improved understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data shows that nicotinamidases are required for growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans regulate lifespan in their respective organisms, consistent with proposed roles in the regulation of NAD+ metabolism and organismal aging. In this manuscript, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, S. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme Disease) and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state kcat values typically exceeding 1 s−1. The Km values for nicotinamide are low and are in the range from 2 – 110 µM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low µM to low nM range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex which is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyse exchange of 18O into the carboxy oxygens of nicotinic acid with 18O-water. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid 18O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion and the intermediacy of a thioester intermediate.
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