ANALYSIS OF SUBSTRATE BINDING BY SITE-SPECIFIC MUTATION

通过位点特异性突变分析底物结合

• 批准号: 3281707
• 负责人: LARRY W COHEN
• 金额: $ 10.04万
• 依托单位: POMONA COLLEGE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-01 至 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3281707
• 关键词: Escherichia coli; chemical structure function; complementary DNA; enzyme structure; enzyme substrate; gel electrophoresis; genetic manipulation; messenger RNA; molecular cloning; nucleic acid sequence; papain; plasmids; point mutation

The objective is to develop an experimental system that will enable 1) the analysis of the effects of amino acid changes on the catalytic mechanism of a eukaryotic enzyme; 2) the demonstration of the potential of directed mutagenesis for re-engineering the properties of an enzyme and 3) the testing and refinement of the equations quantitative structure activity relations (QSAR) currently being used to describe the binding of a ligand to an enzyme. By genetically modifying an already well-studied enzyme (papain) it will be possible to test further the validity of the constants in the equations used to describe the interaction. The specific aim is to transfer the gene for the enzyme papain from the papaya plant into a plasmid of the bacterium E. coli. The sequence of nucleotides in the gene that codes for the amino acid sequence in the protein will then be determined. Using one of the techniques for site-specific mutation, the codes for the amino acid glutamine at position 142 will be changed to that which codes for glycine, or lysine. This will either remove the side chain of the glutamine (as in the change to glycine) which is thought to play a role as a backstop against which the substrate for the enzyme is lodged, or will substitute the lysine side chain that is thought to play the same role in actinidin, a similar enzyme found in Kiwi fruit. The cysteine-25 of the active site will be changed to a serine, as is found in serine proteases and will test the interchangeability of those amino acids in the enzymatic reaction. The altered gene will be sequenced in each case and then positioned in a plasmid so as to get active production of the enzyme by the bacterium. Papain will then be isolated and subsequently subjected to the QSAR analysis to determine what effect the amino acid substitution has had on the constants in the equations. All previous work on enzyme substrate binding has involved varying substituents on a substrate molecule to study enzyme/substrate interaction. This will represent a pioneering study in which the role of portions of the enzyme in substrate binding will be determined by varying the enzyme. At project's end, we hope to be making modifications in the enzyme that restrict the number of substrates accepted. The experimental system will enable refinement of the QSAR equations employed in drug design and will serve as a prototype for studies on the re-engineering of enzymes for maximum therapeutic benefit.

目标是开发一种实验系统，使1) 氨基酸变化对酶催化机理的影响分析 一种真核酶；2)定向酶潜力的论证 用于重新设计酶的性质的突变和3) 定量结构活度方程的检验和改进 关系(QSAR)目前被用来描述配体的结合 一种酶。通过对一种已经研究得很好的酶进行基因改造 (木瓜)将有可能进一步测试常量的有效性 在用来描述相互作用的方程式中。 其具体目的是将木瓜蛋白酶的基因从 将番木瓜植株转化为大肠杆菌的质粒菌。按顺序排列 编码氨基酸序列的基因中的核苷酸 然后将测定蛋白质。使用其中一种技术 定点突变，氨基酸谷氨酰胺的位置密码 142将改为甘氨酸或赖氨酸的代码。这将是 或者去掉谷氨酰胺的侧链(如改为甘氨酸) 它被认为起到了支持底物的作用 因为酶是滞留的，或将取代赖氨酸侧链，即 被认为在猕猴桃中发现的一种类似的酶--放线菌素中起着同样的作用 水果。活性部位的半胱氨酸-25将改变为丝氨酸，因为 在丝氨酸蛋白酶中发现，并将测试这些酶的互换性 酶反应中的氨基酸。 在每种情况下，改变的基因都将被测序，然后定位在 以获得细菌活性产酶的目的。 木瓜酶将被分离出来，随后进行QSAR 分析以确定氨基酸替代对 方程式中的常量。前人对酶底物的研究 结合涉及底物分子上的不同取代基来研究。 酶/底物相互作用。这将是一项开创性的研究 部分酶在底物结合中的作用将是 通过改变酶来决定的。 在项目结束时，我们希望对这种酶进行修改， 限制可接受的底物数量。该实验系统将 使药物设计中使用的QSAR方程得到改进，并将 作为研究酶的再工程的原型 最大限度地提高治疗效益。