Charged Amino Acid Residues Research Articles

Quantum-Chemical Electron Densities of Proteins and of Selected Protein Sites from Subsystem Density Functional Theory.

The ability to calculate accurate electron densities of full proteins or of selected sites in proteins is a prerequisite for a fully quantum-mechanical calculation of protein-protein and protein-ligand interaction energies. Quantum-chemical subsystem methods capable of treating proteins and other biomolecular systems provide a route to calculate the electron densities of proteins efficiently and further make it possible to focus on specific parts. Here, we evaluate and extend the 3-partition frozen-density embedding (3-FDE) scheme [Jacob, C. R.; Visscher, L. J. Chem. Phys.2008, 128, 155102] for this purpose. In particular, we have extended this scheme to allow for the treatment of disulfide bridges and charged amino acid residues and have introduced the possibility to employ more general partitioning schemes. These extensions are tested both for the prediction of full protein electron densities and for focusing on the electron densities of a selected protein site. Our results demonstrate that 3-FDE is a promising tool for the fully quantum-chemical treatment of proteins.

Association of Calcium and Phosphate Ions with Collagen in the Mineralization of Vertebrate Tissues

Among the vertebrate species, collagen is the most abundant protein and is associated with mineralization of their skeleton and dentition in all tissues except enamel. In such tissues, bones, calcifying tendon, dentin, and cementum are comprised principally of type I collagen, which has been proposed as a template for apatite mineral formation. Recent considerations of the interaction between type I collagen and calcium and phosphate ions as the major constituents of apatite have suggested that collagen polypeptide stereochemistry underlies binding of these ions at sites within collagen hole and overlap regions and leads to nucleation of crystals. The concept is fundamental to understanding both normal and abnormal mineralization, and it is reviewed in this article. Given this background, avenues for additional research studies in vertebrate mineralization will also be described. The latter include, for instance, how mineralization events subsequent to nucleation, that is, crystal growth and development, occur and whether they, too, are directed by collagen stereochemical parameters; whether mineralization can be expected in all spaces between collagen molecules; whether the side chains of charged amino acid residues actually point toward and into the hole and overlap collagen spaces to provide putative binding sites for calcium and phosphate ions; and what phenomena may be responsible for mineralization beyond hole and overlap zones and into extracellular tissue regions between collagen structural units. These questions will be discussed to provide a broader understanding of collagen contributions to potential mechanisms of vertebrate mineralization.

Alteration of intersubunit acid–base pair interactions at the quasi-threefold axis of symmetry of Cucumber mosaic virus disrupts aphid vector transmission

In the atomic model of Cucumber mosaic virus (CMV), six amino acid residues form stabilizing salt bridges between subunits of the asymmetric unit at the quasi-threefold axis of symmetry. To evaluate the effects of these positions on virion stability and aphid vector transmissibility, six charged amino acid residues were individually mutated to alanine. All of the six engineered viruses were viable and exhibited near wild type levels of virion stability in the presence of urea. Aphid vector transmissibility was nearly or completely eliminated in the case of four of the mutants; two mutants demonstrated intermediate aphid transmissibility. For the majority of the engineered mutants, second-site mutations were observed following aphid transmission and/or mechanical passaging, and one restored transmission rates to that of the wild type. CMV capsids tolerate disruption of acid–base pairing interactions at the quasi-threefold axis of symmetry, but these interactions are essential for maintaining aphid vector transmissibility.

Molecular Characterization of Human Melanocortin-5 Receptor Ligand–Receptor Interaction

The melanocortin-5 receptor (MC5R) is a subtype receptor of the melanocortin receptor (MCR) family, which is expressed centrally, as well as in a variety of peripheral tissues. MC5R has been implicated in many different physiological fields such as lipid metabolism and exocrine function. However, the specific molecular determinants of MC5R responsible for ligand binding and receptor signaling are currently unknown. The aim of this study is to determine the molecular basis of human MC5R (hMC5R) responsible for ligand binding and receptor signaling. Twenty-four single mutations of hMC5R were created and tested. Our results indicate that (1) substituting charged amino acid residue E92 in transmembrane domain 2 (TM2), aspartic acid 115 (D115) and D119 in TM3, and histidine (H) 257 in TM6 with alanine dramatically reduced NDP-α-MSH binding affinity and receptor signaling and (2) substituting aromatic amino acids phenylalanine (F) 195 in TM5, F254 in TM6, and H276 in TM7 with alanine also significantly decreased NDP-α-MSH binding and receptor activity. Combining pharmacological results and computer modeling, our results suggest that D115 and D119 in TM3, F195 in TM5, and F254 in TM6 may form a binding pocket for NDP-α-MSH binding. Our results provide important information about the structural aspects of hMC5R responsible for ligand binding and receptor signaling.

Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme–substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.

1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A 'nest' comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein-protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner.

DNA Transfection Efficiency of Antimicrobial Peptide as Revealed by Molecular Dynamics Simulation

ABSTRACTThe peptide-DNA complex was investigated by using molecular dynamics simulation to analyze the transfection efficiency of cationic amphipathic peptide. Previously, the cationic peptide, LFampinB, with positively charged amino acid residues of Lysines was used to investigate the orientation and interaction energies for entering the cell though disruption of the endosomal membrane. The same interactions were obtained for N-terminus of the LFampinB peptide with membrane and with plasmid DNA. The N-terminus of LFampinB can bind at minor groove of DNA to make complexation of the peptide with DNA.

Interaction between β-casein micelles and imidazolium-based ionic liquid surfactant

The interaction of β-casein micelles (β-CMs) with imidazolium based ionic liquid (IL) surfactant ([C12mim]Br) has been studied using turbidity, isothermal titration microcalorimetry, fluorescence spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. Below c1, the individual [C12mim]Br monomers bind onto the β-CM shell close to the hydrophobic core to form a β-CM–[C12mim]Br (monomer) complex. The hydrophobic tail of [C12mim]Br leads to a significant decrease in the environmental polarity of β-CM. Just over c1, [C12mim]Br molecules aggregate into micelle-like aggregates on the micellar shell. This leads to the collapse of the N-terminal of β-casein and strengthens the hydrophobicity of the protein molecules, resulting in a more compact structure of β-CM. With a continuous increase in[C12mim]Br concentration, β-CMs associate with each other into a network-like structure. Beyond c3, the net positive charges on the complexes, owing to the binding of more cationic surfactant molecules, lead to redissociation of the complexes, corresponding to the formation of the new nano-sized β-CM–[C12mim]Br complexes. All the β-casein molecules are saturated by [C12mim]Br aggregates above cs, and free [C12mim]Br micelle-like aggregates appear in the bulk phase above critical aggregation concentration (cac). From a combination of experimental results and discussion on various interactions in the β-CM–[C12mim]Br system, the hydrophobic interaction between the hydrophobic tail of ILs and the hydrophobic domain of β-CM, the electrostatic attraction between [C12mim]+ and negative charged amino acid residues on β-CM shell, and the hydrogen bonding between [C12mim]+ and carboxylic moiety on β-CM shell are the main forces for [C12mim]Br binding to β-CM. Modifications in the physicochemical properties of β-CM upon the addition of [C12mim]Br will expand and enhance the overall capabilities and applications of β-CM.

In silico engineering of aggregation-prone recombinant proteins for substrate recognition by the chaperonin GroEL

BackgroundMolecular chaperones appear to have been evolved to facilitate protein folding in the cell through entrapment of folding intermediates on the interior of a large cavity formed between GroEL and its co-chaperonin GroES. They bind newly synthesized or non-native polypeptides through hydrophobic interactions and prevent their aggregation. Some proteins do not interact with GroEL, hence even though they are aggregation prone, cannot be assisted by GroEL for their folding.ResultsIn this study, we have attempted to engineer these non-substrate proteins to convert them as the substrate for GroEL, without compromising on their function. We have used a computational biology approach to generate mutants of the selected proteins by selectively mutating residues in the hydrophobic patch, similar to GroES mobile loop region that are responsible for interaction with GroEL, and compared with the wild counterparts for calculation of their instability and aggregation propensities. The energies of the newly designed mutants were computed through molecular dynamics simulations. We observed increased aggregation propensity of some of the mutants formed after replacing charged amino acid residues with hydrophobic ones in the well defined hydrophobic patch, raising the possibility of their binding ability to GroEL.ConclusionsThe newly generated mutants may provide potential substrates for Chaperonin GroEL, which can be experimentally generated and tested for their tendency of aggregation, interactions with GroEL and the possibility of chaperone-assisted folding to produce functional proteins.

Active Crosstalk with the Microenvironment Leading to Clonal Evolution in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors.

AbstractAbstract 2878We recently demonstrated that intraclonal diversification (ID) in the immunoglobulin (IG) genes of patients with chronic lymphocytic leukemia (CLL) was limited, with the outstanding exception of subset #4 cases (IGHV4–34/IGKV2–30). Subset #4 cases express IgG-switched antigen receptors carrying long VH CDR3s enriched in positively charged amino acid residues (especially arginine), with acidic residues introduced by somatic hypermutation (SHM) in critical positions of both the heavy and light chain variable domains. This group of patients, characterized clinically by an early age at diagnosis and an indolent disease course, exhibited distinctive patterns of intraclonal diversification (ID) within their IG genes. This may be considered as evidence for an ongoing response to active interaction with antigen (Ag), however, the critical question about the precise timing of Ag involvement and its role in clonal evolution remains unknown. To obtain insight into these issues, we conducted a large-scale subcloning study of the IG genes in a total of 514 and 398 subcloned IG heavy and kappa sequences, obtained from overtime samples from 8 subset #4 cases. All non-ubiquitous sequence changes from the germline among subcloned sequences of the same patient from the same timepoint were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences. Overall, all cases carried intraclonally diversified IG genes. Detailed analysis of the topology and characteristics of mutations revealed: i) restricted ID patterns, in the sense of identical mutations at certain VH/VK positions amongst subclones of different cases, e.g. despite glycine at codon 28 (VH CDR1) being mutated in 413/514 (80%) sequences, the only observed substitution was to an acidic residue, ii) ID hotspots, i.e. mutations in certain codons were repeatedly observed during clonal evolution, and iii) predominance of conservative amino acid changes. Furthermore, the analysis of consecutive samples enabled us to trace the diversification of the CLL antibody over time, and describe the level of ID as increasing, decreasing, or complex when a mutation appears, disappears and then re-emerges at a subsequent time point, thereby also revealing which mutations were negatively or positively selected. Consequently, a stepwise accumulation of mutations could be observed with several CMs at an early timepoint becoming ubiquitous mutations, i.e. present in all subcloned sequences of subsequent timepoints. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially, however at later timepoints the minor cluster had often disappeared and hence been selected against. Despite the high intensity of ID, it was remarkable that certain residues such as in the VH FR1 motif responsible for recognizing the I/i NAL epitope remained essentially unaltered (only 6/514 sequences carried alterations at codon 7 VH FR1). In conclusion, this study defines a clear role for Ag selection in the clonal evolution of CLL subset #4. Whilst the critical eliciting Ag cannot be definitively determined, it is tempting to hypothesize that the distinctive modifications introduced by SHM in the stereotyped BcR likely represent a mechanism for negating auto-reactivity mediated by the arginine-rich VH CDR3s and inducing an anergic state which could potentially be re-activated by subsequent (auto)antigenic stimulation as evidenced by ongoing SHM. It remains to be established whether this stimulation is also accompanied by a change in functional status. Disclosures:No relevant conflicts of interest to declare.

A sea anemone polypeptide toxin inhibiting the ASIC3 acid-sensitive channel

Polypeptide toxin pi-AnmTX Hcr 1b-1 with a molecular weight 4537 Da was isolated from the whole body extract of sea anemone by a multistage liquid chromatography. The BLAST search algorithm revealed homology of the novel toxin amino acid sequence to the group of the known sea anemone toxins including BDS and APETx with similarity less then 50%. The toxin pi-AnmTX Hcr 1b-1 inhibited the amplitude of the fast component of integral ASIC3 current in electrophysiological studies on receptors expressed in Xenopus laevis oocytes. The calculated IC50 value was 5.5 +/- 1.0 microM. Among the known polypeptide toxins interacted with ASICs channels, the micro-AnmTX Hcr 1b-1 toxin is the least potent inhibitor that in our opinion correlates with a small amount of charged amino acid residues in its structure.

Bacteriophage‐induced aggregation of oil sands tailings

Very large quantities of tailings are produced as a result of processing oil sands. After the sand particles settle out, a dense stable mixture of clay, silt, water with residual bitumen, salts, and organics called mature fine tailings (MFT) can remain in suspension for decades. Research into developing methods that would allow consolidation and sedimentation of the suspended particles is ongoing. We have studied the ability of a filamentous bacteriophage (called VP12 bearing the peptide DSQKTNPS at the N-terminus of the major coat protein pVIII) to aggregate MFT. To understand the biophysical basis of the aggregation, phage-induced aggregation of diluted MFT was measured at room temperature under varying conditions of pH, salt, detergent. Phage at concentrations of 5.0 × 10(11)/mL to 10(12)/mL induced rapid settling of the diluted MFT. The addition of sodium chloride (10 mM) lowered the concentration of phage required to induce aggregation. Since the non-ionic detergents Triton-X 100 and Tween-20, and the ionic detergent sodium deoxycholate had little effect, hydrophobic interactions do not appear to be a major contributor to the phage-induced aggregation of MFT. However, aggregation was prevented at pH values higher than 9.0 suggesting that positively charged amino acid residues are required for MFT aggregation by phage. Genetic engineering of the pVIII peptide sequence indicated that hydrogen bonding also contributes to phage-induced aggregation. In addition, replacing the basic residue lysine with an alanine in the recombinant peptide of VP12 completely prevented phage-induced aggregation. Three other phage displaying different amino acid sequences but all containing a lysine in the same position had variable aggregation efficiencies, ranging from no aggregation to rapid aggregation. We conclude that not only are the functional groups of the amino acids important, but the conformation that is adopted by the variable pVIII peptide is also important for phage-induced MFT aggregation.

Probing Ion/Molecule Interactions in Aqueous Solutions with Vibrational Energy Transfer

Interactions between model molecules representing building blocks of proteins and the thiocyanate anion, a strong protein denaturant agent, were investigated in aqueous solutions with intermolecular vibrational energy exchange methods. It was found that thiocyanate anions are able to bind to the charged ammonium groups of amino acids in aqueous solutions. The interactions between thiocyanate anions and the amide groups were also observed. The binding affinity between the thiocyanate anion and the charged amino acid residues is about 20 times larger than that between water molecules and the amino acids and about 5-10 times larger than that between the thiocyanate anion and the neutral backbone amide groups. The series of experiments also demonstrates that the chemical nature, rather than the macroscopic dielectric constant, of the ions and molecules plays a critical role in ion/molecule interactions in aqueous solutions.

Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates.

Snake venom contains a diverse array of proteins and polypeptides. Cytotoxins and short neurotoxins are non-enzymatic polypeptide components of snake venom. The three-dimensional structure of cytotoxin and short neurotoxin resembles a three finger appearance of three-finger protein super family. Different family members of three-finger protein super family are employed in diverse biological functions. In this work we analyzed the cytotoxin, short neurotoxin and related non-toxin proteins of other chordates in terms of functional analysis, amino acid compositional (%) profile, number of amino acids, molecular weight, theoretical isoelectric point (pI), number of positively charged and negatively charged amino acid residues, instability index and grand average of hydropathy with the help of different bioinformatical tools. Among all interesting results, profile of amino acid composition (%) depicts that all sequences contain a conserved cysteine amount but differential amount of different amino acid residues which have a family specific pattern. Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins. Different biological system dependent adaptation gives the birth of enriched bio-molecules. Understanding of physicochemical properties of these proteins will help to generate medicinally important therapeutic molecules for betterment of human lives.

Membrane composition influences the topology bias of bacterial integral membrane proteins

Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a “neutral” topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram+ compared to Gram− species and each lipid parameter demonstrated a linear inverse relationship.

Alzheimer’s Disease Amyloid Peptides Interact with DNA, As Proved by Surface Plasmon Resonance

According to the amyloid hypothesis, abnormal processing of the β-amyloid precursor protein in Alzheimer's disease patients increases the production of β-amyloid toxic peptides, which, after forming highly aggregated fibrillar structures, lead to extracellular plaques formation, neuronal loss and dementia. However, a great deal of evidence has point to intracellular small oligomers of amyloid peptides, probably transient intermediates in the process of fibrillar structures formation, as the most toxic species. In order to study the amyloid-DNA interaction, we have selected here three different forms of the amyloid peptide: Aβ1-40, Aβ25-35 and a scrambled form of Aβ25-35. Surface Plasmon Resonance was used together with UV-visible spectroscopy, Electrophoresis and Electronic Microscopy to carry out this study. Our results prove that, similarly to the full length Aβ1-42, all conformations of toxic amyloid peptides, Aβ1-40 and Aβ25-35, may bind DNA. In contrast, the scrambled form of Aβ25-35, a non-aggregating and nontoxic form of this peptide, could not bind DNA. We conclude that although the amyloid-DNA interaction is closely related to the amyloid aggregation proneness, this cannot be the only factor which determines the interaction, since small oligomers of amyloid peptides may also bind DNA if their predominant negatively charged amino acid residues are previously neutralized.

Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA

BackgroundIn interferon-γ-stimulated cells, the dimeric transcription factor STAT1 (signal transducer and activator of transcription 1) recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed GAS (gamma-activated sites). However, the molecular steps that facilitate GAS binding and the subsequent liberation of STAT1 homodimers from these promoter elements are not well understood.ResultsUsing a mutational approach, we identified two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA which efficiently release phospho-STAT1 from DNA. The release of STAT1 dimers from DNA enhances transcriptional activity on both interferon-driven reporter and endogenous target genes. A substitution of either of the two glutamic acid residues broadens the repertoire of putative binding sites on DNA and enhances binding affinity to GAS sites. However, despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the STAT1 DNA-binding mutants show a significantly reduced transcriptional activity upon stimulation of cells with interferon-γ. This reduced transcriptional response may be explained by the deposition of oligomerized STAT1 molecules outside GAS sites.ConclusionsThus, two negatively charged amino acid residues in the DNA-binding domain are engaged in the liberation of STAT1 from DNA, resulting in a high dissociation rate from non-GAS sites as a key feature of STAT1 signal transduction, which positively regulates cytokine-dependent gene expression probably by preventing retention at transcriptionally inert sites.

The charged amino acid residues of two different stators of the flagellar motor in Bacillus subtilis are important for motility

The charged amino acid residues of two different stators of the flagellar motor in Bacillus subtilis are important for motility

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D-enzymes that produce phosphatidic acid-reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.

Transition State Analysis of Enolpyruvylshikimate 3-Phosphate (EPSP) Synthase (AroA)-Catalyzed EPSP Hydrolysis

Proton transfer to carbon atoms is a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. The main catalytic challenge for enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) is protonating the methylene carbon atom of phosphoenolpyruvate, or EPSP, in the reverse reaction. We performed transition state analysis using kinetic isotope effects (KIEs) on AroA-catalyzed EPSP hydrolysis, which also begins with a methylene carbon (C3) protonation, as an analog of AroA's reverse reaction. As part of this analysis, an inorganic phosphate scavenging system was developed to remove phosphate which, though present in microscopic amounts in solution, is ubiquitous. The reaction was stepwise, with irreversible C3 protonation to form an EPSP cation intermediate; that is, an AH(‡)*AN mechanism. The large experimental 3-(14)C KIE, 1.032 ± 0.005, indicated strong coupling of C3 with the motion of the transferring proton. Calculated 3-(14)C KIEs for computational transition state models revealed that the transition state occurs early during C3-H(+) bond formation, with a C3-H(+) bond order of ≈0.24. The observed solvent deuterium KIE, 0.97 ± 0.04, was the lowest observed to date for this type of reaction, but consistent with a very early transition state. The large 2-(14)C KIE reflected an "electrostatic sandwich" formed by Asp313 and Glu341 to stabilize the positive charge at C2. In shifting the transition state earlier than the acid-catalyzed reaction, AroA effected a large Hammond shift, indicating that a significant part of AroA's catalytic strategy is to stabilize the positive charge in the EPSP cation. A computational model containing all the charged amino acid residues in the AroA active site close to the reactive center showed a similar Hammond shift relative to the small transition state models.