2gt2
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(New page: 200px<br /><applet load="2gt2" size="450" color="white" frame="true" align="right" spinBox="true" caption="2gt2, resolution 2.000Å" /> '''Structure of the E....)
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Revision as of 09:15, 21 November 2007
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Structure of the E. coli GDP-mannose mannosyl hydrolase
Overview
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.
About this Structure
2GT2 is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
Reference
X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction., Gabelli SB, Azurmendi HF, Bianchet MA, Amzel LM, Mildvan AS, Biochemistry. 2006 Sep 26;45(38):11290-303. PMID:16981689
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