STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

X射线衍射分析将应用于单晶的一个 一系列半合成牛胰腺核糖核酸酶， 表现出改变的催化效率或底物特异性， 为了进一步界定各种机构所发挥的作用， 活性位点残基在建立催化能力和 这种酶的底物特异性。 父母，完全活跃 半合成酶由以下物质的非共价复合物组成： 残基1-118，通过酶消化天然的 酶，和含有残基III-124的十四肽， 通过化学合成获得。 催化改性的类似物， 研究包括phe-120到leu衍生物（kcat = 13%）， asp-121转化为asn衍生物（kcat = 1.8%），asp-121转化为ala 衍生物（对胞苷环2 '，3'-磷酸的活性= 10%）。 在Leu-120类似物的情况下， 获得了2.0-A的电子密度图，目前正在进行 进一步细化。 asn-121的衍射质量晶体 具有相同空间群且非常接近的导数 与母体结构的晶体同晶。 目前正在努力为ala-121获得合适的晶体 类似物，并且还用于Tyr-120类似物，其表现出双重的 对尿苷环2 '，3'-磷酸的活性增强，但 其他方面都很正常 一种半合成类似物，其中his-119被非常 几乎等排的3-（3-吡唑基）-Ala部分没有活性。 真正的基质扩散到这种类似物的晶体中， 因此，允许直接检查“酶”-底物 配合物 特别令人感兴趣的是一系列的二核苷 磷酸盐底物显示出KCAT值 范围为27至3000秒-1。 如果是相当最终和令人满意的假设， phe-120和asp-121在酶的作用下没有 从单一修改衍生物的审查中出现， 含有吡唑基-119结构和一个或多个 将准备120或121个修改中的其它修改。 如果合适 晶体可以获得这个系列的成员，它应该是 可以确定真正的底物的结构相互作用， 已知酶表现出改变的催化速率或底物 特殊性

X-ray diffraction analysis will be applied to single crystals of a series of semisynthetic bovine pancreatic ribonucleases which exhibit altered catalytic efficiency or substrate specificity, with the intention of delineating further the roles played by various active site residues in establishing the catalytic power and substrate specificity of this enzyme. The parent, fully active semisynthetic enzyme consists of a non covalent complex of residues 1-118, obtained by enzymatic digestion of the native enzyme, and a tetradecapeptide containing residues III-124, obtained by chemical synthesis. Catalytically modified analogs to be studied include the phe-120 to leu derivative (k cat = 13%), the asp-121 to asn derivative (k cat = 1.8%) and the asp-121 to ala derivative (activity against cytidine cyclic 2',3'-phosphate = 10%). In the case of the leu-120 analog, ismorphous crystals have been obtained and a 2.0-A electron density map is presently undergoing further refinement. Diffraction-quality crystals of the asn-121 derivative which have the same space group and are very nearly isomorphous with crystals of the parent structure are in hand. Efforts are underway to obtain suitable crystals for the ala-121 analog, and also for the tyr-120 analog, which exhibits a two-fold enhancement in activity toward uridine cyclic 2',3'-phosphate, but is otherwise normal. The semisyntheitc analog in which his-119 is replaced by the very nearly isosteric 3-(3-pyrazolyl)-ala moiety is devoid of activity. Diffusion of true substrates into crystals of this analog should, therefore, permit direct examination of "enzyme"-substrate complexes. Of particular interest is a series of dinucleoside phosphate substrates that have been shown to exhibit k cat values ranging from 27 to 3000 sec-1 with the native enzyme. If fairly final and satisfactory hypotheses concerning the roles of phe-120 and asp-121 in the action of the enzyme have not emerged from the examination of singly modified derivatives, a series containing both the pyrazolyl-119 structure and one or the other of the 120 or 121 modifications will be prepared. If suitable crystals can be obtained for members of this series, it should be possible to determine structural interactions of true substrates in enzymes know to exhibit altered catalytic rates or substrate specificities.