Gabelli Sandra B, Azurmendi Hugo F, Bianchet Mario A, Amzel L Mario, Mildvan Albert S
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Biochemistry. 2006 Sep 26;45(38):11290-303. doi: 10.1021/bi061239g.
GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k(ex) = 6.6 x 10(4) s(-1)). The structure of the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loop L6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole N epsilon of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metal ions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 10(3.4)-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the beta-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state.
GDP-甘露糖水解酶通过水在糖的C1位进行亲核取代，催化GDP-α-D-己糖转化为GDP和β-D-己糖的水解反应，并伴有构型翻转。两个新的晶体结构（游离酶和酶-底物复合物）、核磁共振（NMR）以及定点诱变数据，与之前报道的酶-产物复合物结构相结合，表明存在一个四阶段催化循环。一个重要的环（L6，残基119 - 125）包含与必需的Mg2+（Gln - 123）、催化碱（His - 124）以及三个阴离子残基结合的配体。由于动态无序，在游离酶的X射线结构中这个环没有有序排列，二维1H - 15N HMQC谱表明了这一点，该谱显示His - 124的咪唑氮共振存在选择性交换加宽（k(ex) = 6.6 x 10(4) s(-1)）。活性较低的Y103F突变体的酶 - Mg2+ - GDP - 甘露糖底物复合物结构显示环L6处于开放构象，而酶 - Mg2+ - GDP产物复合物结构显示环L6处于闭合的“活性”构象。1H - 15N HMQC谱显示His - 124的咪唑Nε未质子化，适合一般碱催化。用亲电性更强的金属离子Mn2+或Co2+取代Mg2+会降低pH对kcat速率曲线中的pKa，表明金属结合水的去质子化部分是限速步骤。H124Q突变使kcat降低10(3.4)倍并基本消除其pH依赖性，而Y103F突变可挽救该突变，Y103F突变使kcat增加23倍并恢复其pH依赖性。挽救的结构基础是Y103F突变将构象平衡转移到开放形式，使环L6移出活性位点，从而允许溶剂中的特定碱氢氧化物直接进入。在所提出的离解过渡态中，该过渡态发生在酶的闭合活性构象中，GDP离去基团的部分负电荷由Mg2+以及使Arg - 37更靠近β - 磷酸的环L2闭合来补偿。当接近类氧化鎓碳正离子过渡态时，甘露糖基C1上正电荷的产生通过将阴离子环L6闭合到活性位点上得到补偿，进一步稳定了过渡态。
