Rate Enhancement in beta-Hydroxyacid Oxidative Decarboxylases

CookEnzyme是一种强大的催化剂，与没有酶的情况下的反应相比，生化反应的速度最高可提高1017倍。为了调节一种酶的活性或设计一种新的催化剂，了解酶是如何催化它们的反应是非常重要的。许多酶通过几个步骤催化它们的反应。因此，在每个单独的化学步骤中，酶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，以确定金属离子依赖的苹果酸酶(ME)和金属离子非依赖的6-磷酸葡萄糖酸脱氢酶(6PGDH)的酸和碱催化、Lewis酸催化和底物结合能的优势。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路径的各个步骤的速率。2.类似物2-脱氧-6PG迅速经历了6PGDH反应的第一步(而不是第二步)。它将被用来将6PGDH反应分成几个步骤。此外，具有受损催化基团的突变6PGDHs和MES将被用于沿着各自酶的催化循环产生缺陷，从而能够分离每个化学步骤。3.利用定点突变技术对ME和6PGDH反应中参与催化和结合的酶残基进行鉴定。对于这两种酶，所有突变体都将根据其结构完整性、反应物的结合以及动力学和化学机制进行表征。

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.