February 28, 2007
A Loop in Action: The Mechanism of the GDP-mannose Mannosyl Hydrolase
S.B. Gabelli1, H.F. Azurmendi2, M.A. Bianchet1, L.M. Amzel1, and A.S. Mildvan2
1Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD;
2Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD
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GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis with inversion of GDP-
-D-hexose to
GDP and 
-D-hexose by nucleophilic substitution at C1 of the hexose. While
the enzyme structure was known, little was understood about the dynamic processes that contribute to the
reaction mechanism. This work identifies the motions in GDPMH involved in enzyme catalysis. We used crystal
structures, and NMR studies, as well as site-directed mutagenesis and molecular modeling of the transition state
to depict four stages in the catalytic cycle of this enzyme: free enzyme, substrate complex, transition state
complex, and product complex.
The three structures of GDPMH – free enzyme, substrate complex, and product complex – suggest that major conformational changes take place during the hydrolysis of GDP-mannose. In the structure of the free enzyme, loop L6 spanning residues 119 to 125 is dynamically disordered (Figure 1A) as indicated by the lack of density in the x-ray structure and by the broadening of the imidazole 15N resonance of His-124 in the HMQC spectrum (Figure 1B). The H124Q mutation decreases kcat 103.4-fold and largely abolishes its pH dependence. The structure of the GDPMH-Mg2+-GDP-mannose complex (substrate complex), carried out using the Y103F mutant (kcat two orders of magnitude lower than that of the wild type enzyme) shows that after binding Mg 2+ and GDP-mannose, the loop becomes ordered but in an open conformation in which the catalytic base, His-124, is 12 Å away from the position required for catalysis (Figure 1A, 2B). This open conformation of the substrate complex appears incompatible with the catalytic requirements of the enzyme.
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In the structure of the product complex, on the other hand, the loop closes onto the catalytic site,
bringing His-124 into the position in which it can act as the catalytic base (closed conformation)
(Figure 1A, 2D). The lack of the tyrosine-OH group could prevent the bound substrate from triggering
the conformational change to that observed in the product complex. However, there is no direct involvement
of the tyrosine-OH in stabilizing the closed conformation. A better explanation is that the partial
positive charge developing on C1 of the mannose during hydrolysis causes loop L6 to close. A closer look
at the structure of the product complex supports this hypothesis, since the product structure contains a
cationic Tris molecule bound in the catalytic site in the position occupied by the substrate mannose in
the substrate complex. The hydroxyl groups of the Tris molecule make hydrogen bonds with side chains of
the same residues that bind the mannose in the substrate complex. Hence, it appears that closing of the
negatively charged catalytic loop L6 (which contains Asp-121, Glu-122, and Asp-125) is driven by the
positive charge that is developing on C1 of the mannose. In the Y103F mutant, the loop L6 does not close
because the hydroxyl of Tyr-103, which makes a hydrogen bond to the leaving oxygen atom of the -phosphate,
is probably required for the charge separation that takes place in the partially dissociative mechanism.
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BEAMLINES
X6A, X25
FUNDING
The National Institutes of Health
PUBLICATION
S.B. Gabelli, H.F. Azurmendi, M.A. Bianchet, L.M. Amzel, and A.S. Mildvan, “X-ray, NMR, and mutational studies of
the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction,” Biochemistry, 45(38), 11290-303, (2006).
FOR MORE INFORMATION
Sandra B. Gabelli, L. Mario Amzel and Albert S. Mildvan
Dept. of Biophysics and Biophysical Chemistry
The Johns Hopkins University
Email: sandra@groucho.med.jhmi.edu