STRUCTURAL ANALYSIS OF SYNTHETICALLY ENGINEERED RNASES

X射线衍射分析将继续应用于单晶 一系列半合成的牛胰腺核糖核酸酶 改变催化效率或底物专一性，目的是 进一步描述天冬氨酸-121和天冬氨酸的作用 苯丙氨酸-120在建立催化能力和底物中的作用 这种酶的专一性。亲本，完全活性的半合成酶 由残基1-118的非共价复合体组成，通过酶法获得 天然酶和含有残基的十四肽的消化 111-124，由化学合成得到。1.8A(R)的精致结构 =20.4)(Martin等人。(1987)J. 比奥尔。化学。262、15930-15938)。如果将asp-121替换为asn或ala，或者 Phe-120被Leu取代，催化效率降低了一个数量级 震级。三种精制的2.0-A结构的催化作用 现在已经获得了有缺陷的类比，但对 酶活性丧失的结构基础被 在所有情况下都发生了多种多样的结构性变化。在 期望更直接的关系将出现在 活性位点配体的存在，我们计划进行结构分析 ASN-121和LEU-120类似物，特别是在 2；-脱氧-2‘-氟尿嘧啶-3’.5‘-腺苷； 过渡态类似物尿苷钒酸；以及产物3‘-胞苷。 一种半合成的模拟物，其中His-119被非常接近的 异构化的3-(3-吡唑基)-丙氨酸部分没有活性。扩散 因此，这种模拟晶体中的真实衬底应该允许 直接检查“酶-底物”复合体。特别感兴趣的 是已经展示的一系列核糖核苷磷酸底物 显示从27到3000秒-1的本机kcat值 酵素。 如果得到的“酶-底物”复合体的结构 吡唑基类似物显示了其他化合物没有观察到的构象特征 活性中心配体，含3-(3-吡唑基)-丙氨酸的双修饰类似物 在119位和亮氨酸在120位或天冬氨酸在121位 将在可能的情况下进行制备、结晶和分析 这些真正的底物。

X-ray diffraction analysis will continue to 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 aspartic acid-121 and phenylalanine-120 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 111-124, obtained by chemical synthesis. A refined structure at 1.8 A (R = 20.4) of the parent complex has been obtained (Martin et al. (1987) J. Biol. Chem. 262, 15930-15938). If asp-121 is replaced by asn or ala, or phe-120 is replaced by leu, catalytic efficiency is reduced an order of magnitude. Refined 2.0-A structures of each of these three catalytically defective analogs have now been obtained, but interpretation of the structural basis for the loss of enzymatic activity is obscured by the multiplicity of structural changes that have occurred in all cases. In the expectation that more straightforward relationships will emerge in the presence of active site ligands, we plan to carry out structural analyses of the asn-121 and leu-120 analogs, in particular in the presence of the virtual substrate, 2;-deoxy-2'-fluorouridilyl-3'.5'-adenosine; the transition state analog, uridine vanadate; and the product, 3'-cytidylate. The semisynthetic 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 ribodinucleoside phosphate substrates that have been shown to exhibit kcat values ranging from 27 to 3000 sec-1 with the native enzyme. If the structures of the "enzyme-substrate" complexes obtained with the pyrazolyl analog reveal conformational features not observed with other active site ligands, double modified analogs containing 3-(3-pyrazolyl)-ala at position 119 and leucine at position 120 or asparagine at position 121 will be prepared, crystallized if possible, and analyzed in the presence of these true substrates.