title: Synergistic inhibitor binding to Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate synthase with both monovalent cations and substrate.
abstract: The inhibitor binding synergy mechanism of the bi-substrate enzyme Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has been investigated with a linkage thermodynamics strategy, involving direct binding experiments of one ligand conducted over a range of concentration of the other. The results demonstrate that binding of the inhibitor glyphosate (GLP) is highly synergistic with both a natural substrate shikimate-3-phosphate (S3P) and activating monovalent cations. The synergy between GLP and S3P binding was determined to be 1600-fold and is in qualitative agreement with previous work on Escherichia coli EPSPS. The binding molar ratios of S3P and GLP were measured as 1.0 and 0.7 per EPSPS, respectively. Monovalent cations that have been shown previously to stimulate S. pneumoniae EPSPS catalytic activity and its inhibition by GLP were found here to exhibit a similar rank-order with respect to their measured GLP binding synergies (ranging from 0 to > or =3000-fold increase in GLP affinity). The cation specificity and the sub-millimolar concentrations where these effects occur strongly suggest the presence of a specific cation binding site. Analytical ultracentrifugation data ruled out GLP-binding synergy mechanisms that derive from, or are influenced by, changes in oligomerization of S. pneumoniae EPSPS. Rather, the data are most consistent with an allosteric mechanism involving changes in tertiary structure. The results provide a quantitative framework for understanding the inhibitor binding synergies in S. pneumoniae EPSPS and implicate the presence of a specific cation binding regulatory site. The findings will help to guide rational design of novel antibiotics targeting bacterial EPSPS enzymes.
authors.affiliation: Department of Anti-Infectives Research, SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.
authors.forename: W
authors.initials: W
authors.lastname: Du
authors.forename: W S
authors.initials: WS
authors.lastname: Liu
authors.forename: D J
authors.initials: DJ
authors.lastname: Payne
authors.forename: M L
authors.initials: ML
authors.lastname: Doyle
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors*; D002151:Calorimetry; D002414:Cations, Monovalent; D002942:Circular Dichroism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D008956:Models, Chemical; D011487:Protein Conformation; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D014461:Ultracentrifugation; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D005998:Glycine; D012765:Shikimic Acid; D000097797:Glyphosate; C042786:shikimic acid-3-phosphate