Rate Enhancement in beta-Hydroxyacid Oxidative Decarboxylases

β-羟基酸氧化脱羧酶的速率增强

• 批准号: 0091207
• 负责人: Paul Cook
• 金额: $ 55万
• 依托单位: University of Oklahoma Norman Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0091207&HistoricalAwards=false
• 关键词: Rate; Enhancement; beta; Hydroxyacid; Oxidative

0091207CookEnzymes are powerful catalysts that increase the rate of a biochemical reaction up to 1017-times compared to the reaction in the absence of enzyme. To modulate an enzyme's activity or to design a new catalyst, it is of import to understand how enzymes catalyze their reaction. A number of enzymes catalyze their reaction via several steps. Thus, overlaying the catalytic role of the enzyme in each of the individual chemical steps is the ability of the enzyme to orchestrate the overall reaction as it proceeds from one step to the other. Research will be conducted to determine information on the advantages derived from acid and base catalysis, Lewis acid catalysis, and substrate binding energy for the metal ion dependent malic enzyme (ME) and the metal ion independent 6-phosphogluconate dehydrogenase (6PGDH). The overall goal will be achieved via the following specific aims. 1. The rates of individual steps along the reaction pathway will be estimated for both enzymes. 2. The analog 2-deoxy-6PG rapidly undergoes the first (but not the second) step of the 6PGDH reaction. It will be used to separate the 6PGDH reaction into individual steps. In addition, mutant 6PGDHs and MEs with impaired catalytic groups will be used to produce defects along the catalytic cycles of the respective enzymes, allowing isolation of each of the chemical steps. 3. Site-directed mutagenesis will be used to identify enzyme residues involved in catalysis and binding in the ME and 6PGDH reactions. For both enzymes, all mutants will be characterized with respect to their structural integrity, binding of reactants, and kinetic and chemical mechanism.

0091207烹饪酶是强大的催化剂，与没有酶的反应相比，它可以将生化反应的速率提高1017倍。 为了调节酶的活性或设计新的催化剂，了解酶如何催化它们的反应是很重要的。 许多酶通过几个步骤催化它们的反应。 因此，酶在每个单独的化学步骤中的催化作用是酶在从一个步骤进行到另一个步骤时协调整个反应的能力。 将进行研究，以确定来自酸和碱催化，刘易斯酸催化，金属离子依赖性苹果酸酶（ME）和金属离子非依赖性6-磷酸葡萄糖酸脱氢酶（6 PGDH）的底物结合能的优势的信息。 总体目标将通过以下具体目标实现。1.将估计两种酶沿反应途径沿着的各个步骤的速率。2.类似物2-脱氧-6PG迅速经历6PGDH反应的第一步（但不是第二步）。 它将用于将6PGDH反应分离为各个步骤。 此外，具有受损催化基团的突变体6PGDH和ME将用于沿着相应酶的催化循环产生沿着缺陷，允许分离每个化学步骤。 3.定点诱变将用于鉴定ME和6PGDH反应中参与催化和结合的酶残基。 对于这两种酶，所有突变体将根据其结构完整性、反应物结合以及动力学和化学机制进行表征。