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首页> 外文期刊>Biochemistry >Characterization of Nicotinamidases: Steady State Kinetic Parameters, Classwide Inhibition by Nicotinaldehydes, and Catalytic Mechanism
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Characterization of Nicotinamidases: Steady State Kinetic Parameters, Classwide Inhibition by Nicotinaldehydes, and Catalytic Mechanism

机译：烟碱酰胺酶的表征：稳态动力学参数，烟醛类的全范围抑制和催化机理。

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摘要

Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. Thesenenzymes are widely distributed across biology, with examples found encoded in the genomes ofMycobacteria,nArchaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Althoughnrecent structural work has improved our understanding of these enzymes, their catalyticmechanismis still notnwell understood. Recent data show that nicotinamidases are required for the growth and virulence of severalnpathogenicmicrobes. The enzymes of Saccharomyces cerevisiae,Drosophilamelanogaster, and Caenorhabditisnelegans regulate life span in their respective organisms, consistent with proposed roles in the regulation ofnNADþ metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidasenenzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for humannpneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparumn(responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts withnsteady state kcat values typically exceeding 1 sn-1n. The Km values for nicotinamide are low and in the range ofn2-110 μM.Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding innthe low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehydenderivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent withnreaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to formna thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidasencan catalyze exchange ofn18nO into the carboxy oxygens of nicotinic acid with H2n18nO. The collected data, alongnwith kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explainsnnicotinamidase and nicotinic acid 18nO exchange chemistry for the S. pneumoniae enzyme involving keyncatalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.
机译：烟碱酰胺酶是将烟酰胺水解成烟酸的代谢酶。酶在整个生物学中广泛分布，发现的例子编码在分枝杆菌，古细菌，真细菌，原生动物，酵母和无脊椎动物的基因组中，但在哺乳动物中没有发现。尽管最近的结构工作改善了我们对这些酶的理解，但对它们的催化机理仍不甚了解。最近的数据表明烟碱酰胺酶是几种致病微生物的生长和毒性所必需的。酿酒酵母，果蝇和果夜蛾的酶调节其各自生物的寿命，与拟议的调节nNADþ代谢和机体衰老的作用一致。在这项工作中，来自秀丽隐杆线虫，Sa。elegans的烟酰胺类酶的稳态动力学参数。据报告有啤酒酵母，肺炎链球菌（引起人类肺炎的病原体），伯氏疏螺旋体（引起莱姆病的病原体）和恶性疟原虫（引起大多数人类疟疾）。烟碱酰胺酶通常是有效的催化剂，其稳态kcat值通常超过1 sn-1n。烟酰胺的Km值很低，在n2-110μM范围内。烟碱醛被确定为这些酶的有效竞争性抑制剂，可在所有测试的烟酰胺酰胺酶中以低微摩尔浓度至低纳摩尔浓度结合。合成了多种烟醛醛衍生物，并在动力学分析中评估了其作为抑制剂。抑制作用与每种酶上普遍保守的催化半胱氨酸与醛羰基碳反应成由保守的氧阴离子孔稳定的甲硫半缩醛复合物的反应一致。肺炎链球菌尼古替丁森能催化n18nO与H2n18nO交换成烟酸的羧基氧。收集的数据，以及几个突变体的动力学分析，使我们能够提出一个催化机理，解释尼古丁酰胺酶和烟酸18nO交换肺炎链球菌酶的化学反应，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体。

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  • 来源
    《Biochemistry》 |2010年第49期|p.10421-10439|共19页
  • 作者

    Jarrod B. French Yana Cen Tracy L. Vrablik Ping Xu Eleanor Allen Wendy Hanna-Rose and Anthony A. Sauve;

  • 作者单位

    ‡Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States,§Tri-InstitutionalProgram in Chemical Biology, Cornell University, 1300 York Avenue, New York, New York 10065, United States,) Department ofPharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10065, United States, and^Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park,Pennsylvania 16802, United States;

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