Synergistic inhibitor binding to Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate synthase with both monovalent cations and substrate

The inhibitor binding synergy mechanism of the bi-substrate enzyme Streptoc occus pneumoniae 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) has be en investigated with a linkage thermodynamics strategy, involving direct bi nding 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-phos phate (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 S 3P 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 c atalytic activity and its inhibition by GLP were found here to exhibit a si milar rank-order with respect to their measured GLP binding synergies (rang ing from 0 to greater than or equal to 3000-fold increase in GLP affinity). The cation specificity and the sub-millimolar concentrations where these e ffects occur strongly suggest the presence of a specific cation binding sit e. Analytical ultracentrifugation data ruled out GLP-binding synergy mechan isms that derive from, or an 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 q uantitative 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.