ANALYSIS OF SUBSTRATE BINDING BY SITE-SPECIFIC MUTATION

目标是开发一个实验系统，使1） 氨基酸变化对催化机理的影响分析 一种真核生物酶; 2）证明了定向酶的潜力， 诱变用于重新工程化酶的性质，和3） 定量结构活性方程的检验和改进 目前用于描述配体结合的定量构效关系（QSAR） 一种酶。 通过基因改造一种已经被充分研究过的酶 这将有可能进一步测试常数的有效性 在用来描述相互作用的方程中。 具体目标是将木瓜蛋白酶的基因从 将木瓜植株的质粒转化为大肠杆菌。杆菌 的序列 在该基因中编码氨基酸序列的核苷酸， 然后将确定蛋白质。 使用其中一种技术， 位点特异性突变，编码位置处的氨基酸谷氨酰胺 142将被改变为编码甘氨酸或赖氨酸的编码。 这将 或者去除谷氨酰胺的侧链（如在甘氨酸的变化中） 其被认为起着支撑的作用， 因为酶被卡住了，或者将取代赖氨酸侧链， 猕猴桃中发现的一种类似的酶， 水果. 活性位点的半胱氨酸-25将变为丝氨酸， 在丝氨酸蛋白酶中发现，并将测试那些 酶促反应中的氨基酸。 将在每种情况下对改变的基因进行测序，然后将其定位在 质粒，使该菌有活性地产生酶。 然后分离木瓜蛋白酶，随后进行QSAR 分析，以确定氨基酸取代对 方程中的常数。 以前所有关于酶底物的工作 结合涉及改变底物分子上的取代基以研究 酶/底物相互作用 这将是一项开创性的研究， 其中酶的部分在底物结合中的作用将是 通过改变酶来确定。 在项目结束时，我们希望对酶进行修改， 限制接受的基质数量。 实验系统将 能够细化药物设计中使用的QSAR方程， 作为研究酶再工程的原型， 最大的治疗效益。

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.