X-ray, NMR, and Mutational Studies of the Catalytic Cycle of the GDP-Mannose Mannosyl Hydrolase Reaction
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- 作者：Sandra B. Gabelli ; Hugo F. Azurmendi ; Mario A. Bianchet ; L. Mario Amzel ; Albert S. Mildvan
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：September 26, 2006
- 年：2006
- 卷：45
- 期：38
- 页码：11290 - 11303
- 全文大小：627K
- 年卷期：v.45,no.38(September 26, 2006)
- ISSN：1520-4995
文摘
GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-
fchars/alpha.gif" BORDER=0>-D-hexose to GDPand fchars/beta2.gif" BORDER=0 ALIGN="middle">-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (freeenzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with thestructure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An importantloop (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 dueto dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selectiveexchange broadening of the imidazole nitrogen resonances of His-124 (kex = 6.6 × 104 s-1). The structureof the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loopL6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole Nfchars/epsilon.gif" BORDER=0 > of His-124 to beunprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metalions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of ametal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 103.4-fold andlargely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold andrestores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shiftsthe conformational equilibrium to the open form moving loop L6 out of the active site, thus permittingdirect 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 GDPleaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to thefchars/beta2.gif" BORDER=0 ALIGN="middle">-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transitionstate is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizingthe transition state.
fchars/alpha.gif" BORDER=0>-D-hexose to GDPand fchars/beta2.gif" BORDER=0 ALIGN="middle">-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (freeenzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with thestructure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An importantloop (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 dueto dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selectiveexchange broadening of the imidazole nitrogen resonances of His-124 (kex = 6.6 × 104 s-1). The structureof the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loopL6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole Nfchars/epsilon.gif" BORDER=0 > of His-124 to beunprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metalions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of ametal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 103.4-fold andlargely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold andrestores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shiftsthe conformational equilibrium to the open form moving loop L6 out of the active site, thus permittingdirect 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 GDPleaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to thefchars/beta2.gif" BORDER=0 ALIGN="middle">-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transitionstate is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizingthe transition state.