Molecular Evolution of Pyridoxal Phoshate Enzymes

描述(由申请人提供)：基因组学、计算生物学和结构生物学在理解蛋白质设计和功能方面取得的重大进展，使人们能够进行直到最近才能进行的关于酶的研究。定向进化或DNA改组将试图转化中间代谢的重要氧化酶苹果酸脱氢酶，以获得结构上相关的酶乳酸脱氢酶的活性，以便将实验室和自然进化的结果进行比较。此外，还将采用同样的技术，试图将酪氨酸氨基转移酶(一种与芳香族和带负电荷的氨基酸反应的酶)的特异性缩小到只涉及后者的活性。参与产生气态激素乙烯的植物酶的定向进化将被用来试图将其转化为氨基转移酶，这是一种非常亲缘的酶。人类2型酪氨酸血症已被证明是由几个突变引起的。为了确定分子缺陷与疾病的确切关系，将对这些突变酶进行表征，包括它们的催化特性和在接近生理条件下的稳定性。人们普遍认识到，很大一部分基因组序列(可能40%)被错误注释。为了底物特异性，将准备并表征不同基因组注释的天冬氨酸和酪氨酸转氨酶的测试集，以试图发现更好的酶基因组注释规则。很少有药物通过破坏蛋白质/蛋白质相互作用来发挥作用。一个主要原因是化验既繁琐又昂贵。正在设计一种质谱学方法，它可以为此目的进行快速高通量分析。

DESCRIPTION (provided by applicant): The major advances in understanding protein design and function arising from genomics, and computational and structural biology, have enabled lines of enquiry to enzymology, that could not have been undertaken until recently. Directed evolution or DNA shuffling will be used to attempt to convert an important oxidation enzyme of intermediary metabolism, malate dehydrogenase, to acquire the activity of a structurally related enzyme, lactate dehydrogenase, in order to compare the results of laboratory with natural evolution. The same technology will be employed, additionally, to attempt to narrow the specificity of tyrosine aminotransferase, an enzyme that reacts with aromatic and negatively charged amino acids, to an activity that engages only the latter class. Directed evolution of a plant enzyme, involved in the production of the gaseous hormone, ethylene, will be used to attempt to convert it into an aminotransferase, a very distantly related enzyme. Human tyrosinemia type 2, has been shown to be caused by a few mutations. These mutant enzymes will be characterized, both with respect to their catalytic properties and their stability under near physiological conditions, in order to define the precise relation of the molecular defect to the disease. It is widely appreciated, that a large fraction of genome sequences (perhaps 40%) has been misannotated. A test set of diverse genome-annotated aspartate and tyrosine aminotransferases, will be prepared and characterized, for substrate specificity, in order to try to discover better rules for genome annotation of enzymes. There are very few drugs that work by disrupting protein/protein interaction. A major reason is that assays are cumbersome and expensive. A mass spectrometric method is being devised, which allows rapid high throughput assays for this purpose.