ENZYME REACTION INTERMEDIATES--A NEW APPROACH

一种结合选角电子核双共振的新方法 (Endor)光谱学结合分子生物学技术选择性地 生物合成的同位素浓缩蛋白质将被应用于确定 A类TEM1β催化活性中心结构 底物水解中的内酰胺酶。硝基-1-自旋标记β-内酰胺 青霉素和头孢菌素的衍生物显示出很高的 催化的专一性和反应性，将被用作光谱 活性中心结构的底物探针。该酶将被分离出来 以过氢藻水解液为培养物的大肠杆菌生长 5~6成熟。营养缺陷型菌株也被适当地改造为过量生产TEM1 将使用β-内酰胺酶在含氢介质中进行生长。 特别是富含同位素的氨基酸。一系列 四种不同的突变体(E104C、E171C、E240C和M272C)将用于 哪些半胱氨酸残基将在6-12A半径内进行工程 氮氧基作为活性中心的战略光谱标记探针 结构。将进一步开发一系列双突变株，每个 含有突变的半胱氨酸残基之一和E166N、R164S或 D179N突变。后两种突变破坏了氢键。 稳定活动位置附近的omega循环，同时E166N呈现 酶去酰化缺陷。米凯利斯情结和 每个突变物种的酰基酶反应中间体将是 低温动力学稳定，可用于Endor光谱分析。除了……之外 R164S和D179N的结构扰动特征 突变，研究将针对确定方向 这两种底物相对于其他关键残留物在 米氏络合物和酰基酶反应中的活性部位 中间体指定蛋白质残基和结构的位置 负责β-内酰胺基团质子化的水分子。这个 Endor距离测量随后将作为约束应用于 相应反应中间体的分子动力学模拟 评估活性中心残基必须如何以构象和 与游离酶的取向相反，承担催化作用 有能力的结构。由于R164S突变提供了抗生素 临床分离株对头孢菌素酶的耐药性，结果也将 确定重要的底物-蛋白质相互作用 对治疗用β-内酰胺酶抑制剂改进设计的理解 目的。

A new approach combining angle selected electron nuclear double resonance (ENDOR) spectroscopy with molecular biological techniques to selectively enrich proteins biosynthetically with isotopes will be applied to determine the catalytically competent active site structure of Class A TEM-1 beta- lactamase in substrate hydrolysis. Nitroxy1 spin-labeled beta-lactam derivatives of penicillin and cephalosporin, shown to exhibit high catalytic specificity and reactivity, will be employed as spectroscopic substrate probes of active site structure. The enzyme will be isolated from E. coli growth on perdeuterated algal hydrolyzate as the culture medium. Auxotrophic strains also suitably engineered to overproduce TEM-1 beta-lactamase will be used for growth on deuterated medium to site specifically incorporate isotopically enriched amino acids. A series of four different mutants (E104C, E171C, E240C, and M272C) will be used into which cysteine residues will be engineered within a 6 - 12 A radius of the nitroxyl group as strategic spectroscopic marker probes of active site structure. A series of double mutants will be further developed, each containing one of the mutant cysteine residues and an E166N, R164S, or D179N mutation. The latter two mutations disrupt the hydrogen bonding stabilizing the omega-loop near the active site while the E166N renders the enzyme deacylation defective. Both the Michaelis complex and the acylenzyme reaction intermediate for each mutant species will be cryokinetically stabilized for ENDOR spectroscopy. In addition to characterizing the structural perturbations due to R164S and D179N mutations, the studies will be directed towards determining the orientation of the two types of substrates with respect to other critical residues in the active site in both Michaelis complex and acylenzyme reaction intermediates to assign the protein residue and location of structured water molecules responsible for protonation of the beta-lactam group. The ENDOR distance measurements will be then applied as constraints in molecular dynamics simulations of the corresponding reaction intermediates to assess how active site residues must rearrange in conformation and orientation from that in the free enzyme to assume a catalytically competent structure. Since the R164S mutation confers antibiotic (cephalosporinase) resistance in clinical isolates, the results will also identify critical substrate-protein interactions that are important to understand for improved design of beta-lactamase inhibitors for therapeutic purposes.