Journal
JOURNAL OF INORGANIC BIOCHEMISTRY
Volume 105, Issue 4, Pages 509-517Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2010.12.012
Keywords
Fructose-1,6-biphosphate aldolase; Giardia lamblia; Zn2+ coordination; Hydroxypyridinone; EXAFS; Inhibitor design
Funding
- NIH [AI059733]
- NSF [CHE-0964806]
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [0946690] Funding Source: National Science Foundation
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Inhibitors of the Giardia lamblia fructose 1,6-bisphosphate aldolase (GlFBPA), which transforms fructose 1,6-bisphosphate (FBP) to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, were designed based on 3-hydroxy-2-pyridone and 1,2-dihydroxypyridine scaffolds that position two negatively charged tetrahedral groups for interaction with substrate phosphate binding residues, a hydrogen bond donor to the catalytic Asp83, and a Zn2+ binding group. The inhibition activities for the GlFBPA catalyzed reaction of FBP of the prepared alkyl phosphonate/phosphate substituted 3-hydroxy-2-pyridinones and a dihydroxypyridine were determined. The 3-hydroxy-2-pyridone inhibitor 8 was found to bind to GlFBPA with an affinity (K-i = 14 mu M) that is comparable to that of FBP (K-m = 2 mu m) or its inert analog TBP (K-i = 1 mu M). The X-ray structure of the GlFBPA-inhibitor 8 complex (2.3 angstrom) shows that 8 binds to the active site in the manner predicted by in silico docking with the exception of coordination with Zn2+. The observed distances and orientation of the pyridone ring O=C-C-OH relative to Zn2+ are not consistent with a strong interaction. To determine if Zn2+ coordination occurs in the GIFBPA-inhibitor 8 complex in solution, EXAFS spectra were measured. A four coordinate geometry comprised of the three enzyme histidine ligands and an oxygen atom from the pyridone ring O=C-C-OH was indicated. Analysis of the Zn2+ coordination geometries in recently reported structures of class II FBPAs suggests that strong Zn2+ coordination is reserved for the enediolate-like transition state, accounting for minimal contribution of Zn2+ coordination to binding of 8 to GlFBPA. (C) 2010 Elsevier Inc. All rights reserved.
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