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α-Carboxyketose synthases, including 3-deoxy-d- arabino heptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N -terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.

3-Deoxy-d- arabino heptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate biosynthetic pathway and is an antimicrobial target. We used an inhibitor-in-pieces approach, based on the previously reported inhibitor DAHP oxime, to screen inhibitor fragments in the presence and absence of glycerol 3-phosphate to occupy the distal end of the active site. This led to DAHP hydrazone, the most potent inhibitor to date, K i = 10 ± 1 nM. Three trifluoropyruvate (TFP)-based inhibitor fragments were efficient inhibitors with ligand efficiencies of up to 0.7 kcal mol -1 /atom compared with 0.2 kcal mol -1 /atom for a typical good inhibitor. The crystal structures showed the TFP-based inhibitors binding upside down in the active site relative to DAHP oxime, providing new avenues for inhibitor development. The ethyl esters of TFP oxime and TFP semicarbazone prevented E. coli growth in culture with IC 50 = 0.21 ± 0.01 and 0.77 ± 0.08 mg mL -1 , respectively. Overexpressing DAHP synthase relieved growth inhibition, demonstrating that DAHP synthase was the target. Growth inhibition occurred in media containing aromatic amino acids, suggesting that growth inhibition was due to depletion of some other product(s) of the shikimate pathway, possibly folate.

NeuB is a bacterial sialic acid synthase used by neuroinvasive bacteria to synthesize N -acetylneuraminate (NeuNAc), helping them to evade the host immune system. NeuNAc oxime is a potent slow-binding NeuB inhibitor. It dissociated too slowly to be detected experimentally, with initial estimates of its residence time in the active site being >47 days. This is longer than the lifetime of a typical bacterial cell, meaning that inhibition is effectively irreversible. Inhibition data fitted well to a model that included a pre-equilibration step with a K i of 36 μM, followed by effectively irreversible conversion to an E*·I complex, with a k 2 of 5.6 × 10 -5 s -1 . Thus, the inhibitor can subvert ligand release and achieve extraordinary residence times in spite of a relatively modest initial dissociation constant. The crystal structure showed the oxime functional group occupying the phosphate-binding site normally occupied by the substrate PEP and the tetrahedral intermediate. There was an ≈10% residual rate at high inhibitor concentrations regardless of how long NeuB and NeuNAc oxime were preincubated together. However, complete inhibition was achieved by incubating NeuNAc oxime with the actively catalyzing enzyme. This requirement for the enzyme to be actively turning over for the inhibitor to bind to the second subunit demonstrated an important role for intersubunit communication in the inhibitory mechanism.

3-Deoxy-d- arabinoheptulosonate 7-phosphate (DAHP) oxime is a transition state mimic inhibitor of bacterial DAHP synthase, with K i = 1.5 μM and a residence time of t R = 83 min. Unexpectedly, DAHP oxime inhibition is competitive with respect to the essential metal ion, Mn 2+ , even though the inhibitor and metal ion do not occupy the same physical space in the active site. This is problematic because DAHP synthase is activated by multiple divalent metal cations, some of which have significant intracellular concentrations and some of which dissociate slowly. The nature of DAHP oxime's competition with the metal ion was investigated. Inhibition shifted from metal-competitive at physiological pH to metal-noncompetitive at pH > 8.7 in response to deprotonation of the Cys61 side chain. The modes of inhibition of DAHP synthase mutants and inhibitor fragments demonstrated that metal-competitive inhibition arose from interactions between Mn 2+ , DAHP oxime's O4 hydroxyl group, and the Cys61 and Asp326 side chains. The majority of potent DAHP synthase inhibitors in the literature possess a 4-hydroxyl group. Removing it could avoid metal-competitive inhibition and avoid them being outcompeted by metal ions in vivo.

3-Deoxy-d- manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the first step of lipopolysaccharide biosynthesis, namely condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P), to produce KDO8P. We have characterized Campylobacter jejuni KDO8P synthase and its inhibition by KDO8P oxime. It was metal-dependent and homotetrameric and followed a rapid equilibrium sequential ordered ter ter kinetic mechanism in which Mn 2+ bound first, followed by PEP and then A5P. It was inhibited by KDO8P oxime, an analogue of 3-deoxy-d- arabino-heptulosonate 7-phosphate (DAHP) oxime, a transition-state mimic of DAHP synthase. Inhibition was uncompetitive-like with respect to Mn 2+ and competitive with respect to PEP and A5P. It displayed both fast-binding inhibition ( K i = 10 μM) and slow-binding inhibition ( K i * = 0.57 μM). The residence times on the enzyme ( t R ) ranged from 27 min in the absence of free inhibitor to 69 h with excess inhibitor. The dependence of t R on the free inhibitor concentration suggested intersubunit communication within the homotetramer between high- and low-affinity sites. This confirms the generality of the oxime functional group, a small, neutral phosphate bioisostere, as an α-carboxyketose synthase inhibitor and highlights the challenge that intersubunit communication poses to effective inhibition.

Introduction: Deferoxamine (DFO) is a siderophore that bacteria use to scavenge iron and could serve as a targeting vector to image bacterial infection where current techniques have critical limitations. [ 67 Ga]-DFO, which is a mimetic of the corresponding iron complex, is taken up by bacteria in culture, however in vivo it clears too rapidly to allow for imaging of infection. In response, we developed several new DFO derivatives to identify those that accumulate in bacteria, and at sites of infection, and that could potentially have improved pharmacokinetics., Methods: A library of DFO derivatives was synthesized by functionalizing the terminal amine group of DFO using three different carbamate-forming reactions. Uptake of [ 67 Ga]-DFO and the 67 Ga-labeled derivatives by bacteria and the biodistribution of lead compounds were studied., Results: 67 Ga-labeled DFO derivatives were prepared and isolated in >90% radiochemical yield and >95% radiochemical purity. The derivatives had significant but slower uptake rates in Staphylococcus aureus than [ 67 Ga]-DFO (6% to 60% of the control rate), with no uptake for the most lipophilic derivatives. Biodistribution studies in mice with a S. aureus infection in one thigh revealed that the ethyl carbamate derivative had an excellent infected-to-non-infected ratio (11:1), but high non-specific localization in the gall bladder, liver and small intestine., Conclusions: The work reported shows that it is possible to functionalize DFO-type siderophores and retain active uptake of the 67 Ga-labeled complexes by bacteria. Novel 67 Ga-labeled DFO derivatives were specifically taken up by S. aureus and selected derivatives demonstrated in vivo localization at sites of infection. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: 67 Ga-labeled DFO derivatives were actively transported by bacteria using the iron-siderophore pathway, suggesting that it is possible to develop siderophore-based radiopharmaceuticals for imaging bacterial infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with K i = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(K M,Mn K M,PEP K M,E4P /k cat ) versus log(K i ), revealed a slope, m, of 0.34, with r 2 = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.

A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with 99mTc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The 99mTc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability in vitro and in vivo. A trans-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with 99mTc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with 99mTc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control 99mTc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting Staphylococcus aureus infection in vitro and in vivo. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the 99mTc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies., Competing Interests: The authors have declared that no competing interests exist.

3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn 2+ , binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with K i = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, t R , of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The k cat value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent K M,E4P value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic.

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.

Proton transfer to carbon represents a significant catalytic challenge because of the large intrinsic energetic barrier and the frequently unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs) were measured for acid-catalyzed hydrolysis of the enol ether functionality of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog of the EPSP synthase (AroA) reaction. The large solvent deuterium KIE demonstrated that protonating C3 was the rate-limiting step, and the lack of solvent hydron exchange into EPSP demonstrated that protonation was irreversible. The reaction mechanism was stepwise, with C3, the methylene carbon, being protonated to form a discrete oxacarbenium ion intermediate before water attack at the cationic center, that is, an AH(‡)*AN (or AH(‡) + AN) mechanism. The calculated 3-(14)C and 3,3-(2)H2 KIEs varied as a function of the extent of proton transfer at the transition state, as reflected in the C3-H(+) bond order, nC3-H+. The calculated 3-(14)C KIE was a function primarily of C3 coupling with the movement of the transferring proton, as reflected in the reaction coordinate contribution ((light)ν(‡)/(heavy)ν(‡)), rather than of changes in bonding. Coupling was strongest in early and late transition states, where the reaction coordinate frequency was lower. The other calculated (14)C and (18)O KIEs were more sensitive to interactions with counterions and solvation in the model structures than nC3-H+. The KIEs revealed a moderately late transition state with significant oxacarbenium ion character and with a C3-H(+) bond order ≈0.6.

This work introduces an integrated microfluidic device for measuring rapid H/D exchange (HDX) in proteins. By monitoring backbone amide HDX on the millisecond to low second time scale, we are able to characterize conformational dynamics in weakly structured regions, such as loops and molten globule-like domains that are inaccessible in conventional HDX experiments. The device accommodates the entire MS-based HDX workflow on a single chip with residence times sufficiently small (ca. 8 s) that back-exchange is negligible (≤5%), even without cooling. Components include an adjustable position capillary mixer providing a variable-time labeling pulse, a static mixer for HDX quenching, a proteolytic microreactor for rapid protein digestion, and on-chip electrospray ionization (ESI). In the present work, we characterize device performance using three model systems, each illustrating a different application of 'time-resolved' HDX. Ubiquitin is used to illustrate a crude, high throughput structural analysis based on a single subsecond HDX time-point. In experiments using cytochrome c, we distinguish dynamic behavior in loops, establishing a link between flexibility and interactions with the heme prosthetic group. Finally, we localize an unusually high 'burst-phase' of HDX in the large tetrameric enzyme DAHP synthase to a 'molten globule-like' region surrounding the active site.

MurAs (enolpyruvyl-UDP-GlcNAc synthases) from pathogenic bacteria such as Borrelia burgdorferi (Lyme disease) and tuberculosis are fosfomycin resistant because an Asp-for-Cys substitution prevents them from being alkylated by this epoxide antibiotic. Previous attempts to characterize naturally Asp-containing MurAs have resulted in no protein or no activity. We have expressed and characterized His-tagged Lyme disease MurA (Bb&#95;MurA(H6)). The protein was most soluble at high salt concentrations but maximally active around physiological ionic strength. The steady-state kinetic parameters at pH 7 were k(cat) = 1.07 ± 0.03 s(-1), K(M,PEP) = 89 ± 12 μM, and K(M,UDP-GlcNAc) = 45 ± 7 μM. Mutating the active site Asp to Cys, D116C, caused a 21-fold decrease in k(cat) and rendered the enzyme fosfomycin sensitive. The pH profile of k(cat) was bell-shaped and centered around pH 5.3 for Bb&#95;MurA(H6), with pK(a1) = 3.8 ± 0.2 and pK(a2) = 7.4 ± 0.2. There was little change in pK(a1) with the D116C mutant, 3.5 ± 0.3, but pK(a2) shifted to >11. This demonstrated that the pK(a2) of 7.4 was due to D116, almost 3 pH units above an unperturbed carboxylate, and that it must be protonated for activity. This supports D116's proposed role as a general acid/base catalyst. As fosfomycin does not react with simple thiols, nor most protein thiols, the reactivity of D116C with fosfomycin, combined with the strongly perturbed pK(a2) for D116, strongly implies an unusual active site environment and a chemical role in catalysis for Asp/Cys. There is also good evidence for C115 having a role in product release. Both roles may be operative for both Asp- and Cys-containing MurAs.

MurA (enolpyruvyl UDP-GlcNAc synthase) catalyzes the first committed step in peptidoglycan biosynthesis. In this study, MurA-catalyzed breakdown of its tetrahedral intermediate (THI), with a k(cat)/K(M) of 520 M(-1) s(-1), was far slower than the normal reaction, and 3 x 10(5)-fold slower than the homologous enzyme, AroA, reacting with its THI. This provided kinetic evidence of slow binding and a conformationally constrained active site. The MurA cocrystal structure with UDP-N-acetylmuramic acid (UDP-MurNAc), a potent inhibitor, and phosphite revealed a new "staged" MurA conformation in which the Arg397 side chain tracked phosphite out of the catalytic site. The closed-to-staged transition involved breaking eight MurA.ligand ion pairs, and three intraprotein hydrogen bonds helping hold the active site loop closed. These were replaced with only two MurA.UDP-MurNAc ion pairs, two with phosphite, and seven new intraprotein ion pairs or hydrogen bonds. Cys115 appears to have an important role in forming the staged conformation. The staged conformation appears to be one step in a complex choreography of release of the product from MurA.

Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) catalyzes the sixth step in aromatic amino acid biosynthesis. It forms EPSP from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) in an addition/elimination reaction that proceeds through a tetrahedral intermediate. In spite of numerous mechanistic studies, the catalytic roles of specific amino acid residues remain an open question. Recent experimental evidence for cationic intermediates or cationic transition states, and a consideration of the catalytic imperative, have guided this study on the catalytic roles of Lys22 (K22), Asp313 (D313), and Glu341 (E341). Steady-state and pre-steady-state kinetics and protein stability studies showed that mutations of D313 and E341 caused k(cat) to decrease up to 30,000-fold and 76,000-fold, respectively, while the effects on K(M) were modest, never more than 40-fold. Thus, both are identified as catalytic residues. In an active site that is overwhelmingly positively charged, the D313 and E341 side chains are positioned to form an "electrostatic sandwich" around the positive charge at C2 in cationic intermediates/transition states, stabilizing them and thereby promoting catalysis. Mutation of K22 showed large effects on K(M,S3P) (100-fold), K(M,PEP) (>760-fold), and up to 120-fold on k(cat). Thus, K22 had roles in both substrate-binding and transition-state stabilization. These results support the identification of E341 and K22 as general acid/base catalytic residues.

The transition state (TS) structure of MutY-catalyzed DNA hydrolysis was solved using multiple kinetic isotope effect (KIE) measurements. MutY is a base excision repair enzyme which cleaves adenine from 8-oxo-G:A mismatches in vivo, and also from G:A mismatches in vitro. TS analysis of G:A-DNA hydrolysis revealed a stepwise S(N)1 (D(N)*A(N)(double dagger)) mechanism proceeding through a highly reactive oxacarbenium ion intermediate which would have a lifetime in solution of <10(-10) s. C-N bond cleavage is reversible; the N-glycoside bond breaks and reforms repeatedly before irreversible water attack on the oxacarbenium ion. KIEs demonstrated that MutY uses general acid catalysis by protonating N7. It enforces a 3'-exo sugar ring conformation and other sugar ring distortions to stabilize the oxacarbenium ion. Combining the experimental TS structure with the previously reported crystal structure of an abortive Michaelis complex elucidates the step-by-step catalytic sequence.

Multiple kinetic isotope effects (KIEs) on deoxyadenosine monophosphate (dAMP) hydrolysis in 0.1 M HCl were used to determine the transition state (TS) structure and probe its intrinsic reactivity. The experimental KIEs revealed a stepwise (SN1) mechanism, with a discrete oxacarbenium ion intermediate. This is the first direct evidence for the deoxyribosyl oxacarbenium ion in solution. In 50% methanol/0.1 M HCl the products were deoxyribose 5-phosphate (dRMP) and alpha- and beta-methyl dRMP. The alpha-Me-dRMP/beta-Me-dRMP ratio was 8.5:1. Assuming that a free oxacarbenium ion is equally susceptible to nucleophilic attack on either face, this indicated that approximately 20% proceeded through a solvent-separated ion pair complex, or free oxacarbenium ion, a DN+AN mechanism, while approximately 80% of the reaction proceeded through a contact ion pair complex. The oxacarbenium ion lifetime was estimated at 10(-11)-10(-10) s. Computational transition states were found for ANDN, DN*AN, DN*AN, and DN+AN mechanisms using hybrid density functional theory calculations. After taking into account 20% of DN+AN, there was an excellent match of calculated to experimental KIEs for 80% of the reaction having a DN*AN mechanism. That is, C-N bond cleavage is reversible, with dAMP and the {oxacarbenium ion*adenine} complex in equilibrium. The first irreversible step is water attack on the oxacarbenium ion. The calculated 1'-14C KIE for a stepwise mechanism with irreversible C-N bond cleavage (DN*AN) was 1.052, in the range previously associated only with ANDN transition states, and close to the calculated ANDN value, 1.059. The 1'-14C KIE was strongly dependent on the adenine protonation state.

Enolpyruvylshikimate-3-phosphate synthase (AroA, also called EPSP synthase) is a carboxyvinyl transferase involved in aromatic amino acid biosynthesis, forming EPSP from shikimate 3-phosphate and phosphoenolpyruvate. Upon extended incubation, EPSP ketal, a side product, forms by intramolecular nucleophilic addition of O4 to C2' of the enolpyruvyl group. The catalytic significance of this reaction was unclear, as it was initially proposed to arise from nonenzymatic breakdown of tetrahedral intermediate that had dissociated from AroA. This study shows that EPSP ketal formed in AroA's active site, not nonenzymatically, by demonstrating its formation in the presence of excess AroA. It formed both in the normal reaction and during AroA-catalyzed EPSP hydrolysis. In addition, nonenzymatic EPSP hydrolysis was studied to elucidate the catalytic imperative for enolpyruvyl reactions. Hydrolysis was acid-catalyzed, with a rate enhancement of >5 x 10(8)-fold. There was no detectable EPSP breakdown after 16 days at 90 degrees C in 1 M KOH, a solution that is 1000-fold more nucleophilic than neutral aqueous solutions. Thus, an unactivated enolpyruvyl group is not susceptible to nucleophilic attack. Enzymatic EPSP ketal formation therefore requires enolpyruvyl activation through protonation of C3' to form either a cationic intermediate or a highly cation-like transition state. Forming an EPSP cation requires the investment of considerable catalytic power by AroA. Such an intermediate is a potential target motif for inhibitor design.

The role in catalysis of phosphate with AroA (enolpyruvyl shikimate 3-phosphate synthase) and MurA (enolpyruvyl UDP-GlcNAc synthase) was probed using phosphate analogues and an AroA mutant. Phosphate, the second reaction product, increases the reactivity of the enolpyruvyl products (EP-OR's) approximately 10(5)-fold in the reverse reaction, forming phosphoenolpyruvate and R-OH (shikimate 3-phosphate or UDP-GlcNAc). Phosphate is intrinsically unreactive with EP-OR, raising the question of how AroA and MurA promote EP-OR reactivity. Eleven phosphate analogues were examined. All those with tetrahedral geometries bound with AroA, except sulfate, while no nontetrahedral analogues did. Arsenate, vanadate, and fluorophosphate caused reactions of AroA and MurA with EP-OR's, yielding pyruvate and R-OH. Their k(cat)/K(M) values relative to phosphate were similar for both enzymes, ca. 100-fold worse for arsensate, 200-fold worse for vanadate, and 5000-fold worse for fluorophosphate, implying similar interactions with both enzymes. Examination of the arsenate-promoted reactions using [3'-(3)H]EP-OR's, (2)H(2)O, and H(2)(18)O provided evidence of an arseno-tetrahedral intermediate, analogous to the natural tetrahedral intermediate, proceeding to arsenoenolpyruvate, which spontaneously broke down to pyruvate and arsenate. The only physicochemical property that appeared to be essential for reactivity of the analogues was the presence of a proton. Titration of the intrinsic tryptophan fluorescence of the weakly active AroA mutant, Asp313Ala (D313A), demonstrated a fluorescence decrease upon enolpyruvyl shikimate 3-phosphate (EPSP) binding, and a further decrease upon binding of phosphate or arsenate to AroA&#95;D313A.EPSP, suggesting a further conformational change. We are hopeful that understanding enzyme-phosphate interactions will make it possible to design inhibitors that can use the high endogenous phosphate concentration in bacteria to enhance inhibitor binding.

A recent study of phosphate monoesters that broke down exclusively through C-O bond cleavage and whose reactivity was unaffected by protonation of the nonbridging oxygens (Byczynski et al. J. Am. Chem. Soc. 2003, 125, 12541) raised several questions about the reactivity of phosphate monoesters, R-O-P(i). Potential catalytic strategies, particularly with regard to selectively promoting C-O or O-P bond cleavage, were investigated computationally through simple alkyl and aryl phosphate monoesters. Both C-O and O-P bonds lengthened upon protonating the bridging oxygen, R-O(H(+))-P(i), and heterolytic bond dissociation energies, DeltaH(C)(-)(O) and DeltaH(O)(-)(P), decreased. Which bond will break depends on the protonation state of the phosphoryl moiety, P(i), and the identity of the organosubstituent, R. Protonating the bridging oxygen when the nonbridging oxygens were already protonated favored C-O cleavage, while protonating the bridging oxygen of the dianion form, R-O-PO(3)(2)(-), favored O-P cleavage. Alkyl R groups capable of forming stable cations were more prone to C-O bond cleavage, with tBu > iPr > F(2)iPr > Me. The lack of effect on the C-O cleavage rate from protonating nonbridging oxygens could arise from two precisely offsetting effects: Protonating nonbridging oxygens lengthens the C-O bond, making it more reactive, but also decreases the bridging oxygen proton affinity, making it less likely to be protonated and, therefore, less reactive. The lack of effect could also arise without bridging oxygen protonation if the ratio of rate constants with different protonation states precisely matched the ratio of acidity constants, K(a). Calculations used hybrid density functional theory (B3PW91/6-31++G) methods with a conductor-like polarizable continuum model (CPCM) of solvation. Calculations on Me-phosphate using MP2/aug-cc-pVDZ and PBE0/aug-cc-pVDZ levels of theory, and variations on the solvation model, confirmed the reproducibility with different computational models.

Purified recombinant MurA (enolpyruvyl-UDP-GlcNAc synthase) overexpressed in Escherichia coli had significant amounts of UDP-MurNAc (UDP-N-acetylmuramic acid) bound after purification. UDP-MurNAc is the product of MurB, the next enzyme in peptidoglycan biosynthesis. About 25% of MurA was complexed with UDP-MurNAc after five steps during purification that should have removed it. UDP-MurNAc isolated from MurA was identified by mass spectrometry, NMR analysis, and comparison with authentic UDP-MurNAc. Subsequent investigation showed that UDP-MurNAc bound to MurA tightly, with K(d,UDP)(-)(MurNAc) = 0.94 +/- 0.04 microM, as determined by fluorescence titrations using ANS (8-anilino-1-naphthalenesulfonate) as an exogenous fluorophore. UDP-MurNAc binding was competitive with ANS and phosphate, the second product of MurA, and it inhibited MurA. The inhibition patterns were somewhat ambiguous, likely being competitive with the substrate PEP (phosphoenolpyruvate) and either competitive or noncompetitive with respect to the substrate UDP-GlcNAc (UDP-N-acetylglucosamine). These results indicate a possible role for UDP-MurNAc in regulating the biosynthesis of nucleotide precursors of peptidoglycan through feedback inhibition. Previous studies indicated that UDP-MurNAc binding to MurA was not tight enough to be physiologically relevant; however, this was likely an artifact of the assay conditions.

Kinetic isotope effects (KIEs) were measured for methyl glucoside (4) hydrolysis on unlabeled material by NMR. Twenty-eight (13)C KIEs were measured on the acid-catalyzed hydrolysis of alpha-4 and beta-4, as well as enzymatic hydrolyses with yeast alpha-glucosidase and almond beta-glucosidase. The 1-(13)C KIEs on the acid-catalyzed reactions of alpha-4 and beta-4, 1.007(2) and 1.010(6), respectively, were in excellent agreement with the previously reported values (1.007(1), 1.011(2): Bennet and Sinnott, J. Am. Chem. Soc. 1986, 108, 7287). Transition state analysis of the acid-catalyzed reactions using the (13)C KIEs, along with the previously reported (2)H KIEs, confirmed that both reactions proceed with a stepwise D(N)A(N) mechanism and showed that the glucosyl oxocarbenium ion intermediate exists in an E(3) sofa or (4)H(3) half-chair conformation. (13)C KIEs showed that the alpha-glucosidase reaction also proceeded through a D(N)*A(N) mechanism, with a 1-(13)C KIE of 1.010(4). The secondary (13)C KIEs showed evidence of distortions in the glucosyl ring at the transition state. For the beta-glucosidase-catalyzed reaction, the 1-(13)C KIE of 1.032(1) demonstrated a concerted A(N)D(N) mechanism. The pattern of secondary (13)C KIEs was similar to the acid-catalyzed reaction, showing no signs of distortion. KIE measurement at natural abundance makes it possible to determine KIEs much more quickly than previously, both by increasing the speed of KIE measurement and by obviating the need for synthesis of isotopically labeled compounds.

The mechanisms of nonenzymatic breakdown of the tetrahedral intermediates (THIs) of the carboxyvinyl transferases MurA and AroA were examined in order to illuminate the interplay between the inherent reactivities of the THIs and the enzymatic strategies used to promote catalysis. THI degradation was through phosphate departure, with C-O bond cleavage. It was acid catalyzed and dependent on the protonation state of the carboxyl of the alpha-carboxyketal phosphate functionality, with ionizations at pK(a) = 3.2 +/- 0.1 and 4.3 +/- 0.1 for MurA and AroA THIs, respectively. The solvent deuterium kinetic isotope effect for MurA THI at pL 2.0 was 1.3 +/- 0.4, consistent with general acid catalysis. The pK(a)'s suggested intramolecular general acid catalysis through protonation of the bridging oxygen of the phosphate, though H(3)O(+) catalysis was also possible. The product distribution varied with pH. The dominant breakdown products were pyruvate + phosphate + R-OH (R-OH = UDP-GlcNAc or shikimate 3-phosphate) at all pH's, particularly low pH. At higher pH's, increasing proportions of ketal, arising from intramolecular substitution of phosphate by the adjacent hydroxyl and the enolpyruvyl products of phosphate elimination were observed. With MurA THI, the product distribution fitted to pK(a)'s 1.6 and 6.2, corresponding to the expected pK(a)'s of a phosphate monoester. C-O bond cleavage was demonstrated by the lack of monomethyl [(33)P]phosphate formed upon degrading MurA [(33)P]THI in 50% methanol. General acid catalysis through the bridging oxygen is consistent with the location of the previously proposed general acid catalyst for THI breakdown in AroA, Lys22.

MutY, a DNA repair enzyme, is unusual in that it binds exceedingly tightly to its products after the chemical steps of catalysis. Until now it was not known whether the product being released in the rate-limiting step was DNA, adenine, or both. MutY hydrolyzes adenine from 8-oxo-G:A (OG:A) base pair mismatches as the first step in the base excision repair pathway, as well as from G:A mismatches. The products are adenine and DNA containing an apurinic (AP) site. Tight product binding may have a physiological role in preventing further damage at the OG:AP site. We developed a rate assay using [8-14C]adenine in OG:A or G:A mismatches that distinguishes between adenine hydrolysis and adenine release. [8-14C]Adenine was released quickly from the MutY.AP-DNA.[8-14C]adenine complex, with a rate constant greater than 5 min-1. This was much faster than the rate-limiting step, at 0.006-0.015 min-1. Gel retardation experiments showed that AP-DNA release was very slow, consistent with it being the rate-limiting step. Thus, the kinetic mechanism involves fast adenine release after hydrolysis followed by rate-limiting AP-DNA release. Adenine appears to be buried deep in the protein.DNA interface, but there is enough flexibility or open space for it to dissociate from the MutY.APDNA.adenine complex. These results have implications for the catalytic mechanism of MutY.

AroA (EPSP synthase) catalyzes carboxyvinyl transfer through addition of shikimate 3-phosphate (S3P) to phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (THI), followed by phosphate elimination to give enolpyruvylshikimate 3-phosphate (EPSP). A novel approach, partitioning analysis, was used to elucidate the roles of catalytic residues in each step of the reaction. Partitioning analysis involved trapping and purifying [1-(14)C]THI, degrading it with AroA, and quantitating the products. Wild-type AroA gave a partitioning factor, f(PEP) = 0.25 +/- 0.02 at pH 7.5, where f(PEP) = [[1-(14)C]PEP]/([[1-(14)C]PEP] + [[1-(14)C]EPSP]). Eighteen mutations were made to 14 amino acids to discover which residues preferentially catalyzed either the addition or the elimination step. Mutating a residue catalyzing one step (e.g., addition) should change f(PEP) to favor the opposite step (e.g., elimination). No mutants caused large changes in f(PEP), with experimental values from 0.07 to 0.41. This implied that there are no side chains that catalyze only addition or elimination, which further implied that the same residues are general acid/base catalysts in both forward and reverse THI breakdown. Only Lys22 (protonating S3P hydroxyl or phosphate) and Glu341 (deprotonating C3 of PEP) are correctly situated in the active site. In the overall reaction, Lys22 would act as a general base during addition, while Glu341 would act as a general acid. Almost half of the mutations (eight of 18) caused a >1000-fold decrease in specific activity, demonstrating that a large number of residues are important for transition state stabilization, "ensemble catalysis", in contrast to some enzymes where a single amino acid can be responsible for up to 10(8)-fold catalytic enhancement.

BEBOVA-based TS determination has been very successful in elucidating enzyme mechanisms at a level of detail that would be otherwise inaccessible. The resulting TS structures have been used successfully as the basis for designing TS mimics as enzyme inhibitors with dissociation constants to 10(-11) M. The structure interpolation approach has systematized the process of finding a TS, increasing both the speed and the accuracy of TS determination. The combination of information from several TSs into a unified model increases the accuracy of the process significantly and results in an extremely sensitive probe of changes in TS with varying reaction conditions (i.e., enzymatic vs nonenzymatic reactions, different enzymes, or different nucleophiles). The TS determination process is summarized in Fig. 15.

Pertussis toxin ADP-ribosylates a specific Cys side chain in the alpha-subunit of several G-proteins. Recombinant Gialpha1-subunits were rapidly ADP-ribosylated in the absence of betagamma-subunits, with a Km of 800 microM and a kcat of 40 min-1. Addition of betagamma-subunits decreases Km to 0.3 microM with little change of kcat. Kinetic isotope effects established the transition-state structure for ADP-ribosylation of Gialpha1 subunits. The transition state is dissociative, with a 2.1 A bond to the nicotinamide leaving group and a bond of 2.5 A to the sulfur nucleophile. The nucleophilic participation of Gialpha1 at the transition state is greater than that for water in the hydrolysis of NAD+by pertussis toxin. Crystal structures for Gialpha1 show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered Gialpha1 conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.

Diphtheria toxin (DTA) uses NAD(+) as an ADP-ribose donor to catalyze the ADP-ribosylation of eukaryotic elongation factor 2. This inhibits protein biosynthesis and ultimately leads to cell death. In the absence of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD(+) to ADP-ribose and nicotinamide, a reaction that can be exploited to measure kinetic isotope effects (KIEs) of isotopically labeled NAD(+)s. Competitive KIEs were measured by the radiolabel method for NAD(+) molecules labeled at the following positions: 1-(15)N = 1.030 ± 0.004, 1'-(14)C = 1.034 ± 0.004, (1-(15)N,1'-(14)C) = 1.062 ± 0.010, 1'-(3)H = 1.200 ± 0.005, 2'-(3)H = 1.142 ± 0.005, 4'-(3)H = 0.990 ± 0.002, 5'-(3)H = 1.032 ± 0.004, 4'-(18)O = 0.986 ± 0.003. The ring oxygen, 4'-(18)O, KIE was also measured by whole molecule mass spectrometry (0.991 ± 0.003) and found to be within experimental error of that measured by the radiolabel technique, giving an overall average of 0.988 ± 0.003. The transition state structure of NAD(+) hydrolysis was determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational analysis to predict the KIEs of the trial structures. The predicted KIEs matched the experimental ones for a concerted, highly oxocarbenium ion-like transition state. The residual bond order to the leaving group was 0.02 (bond length = 2.65 Å), while the bond order to the approaching nucleophile was 0.03 (2.46 Å). This is an A(N)D(N) mechanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the active site cleft of the X-ray crystallographic structure of DTA highlighted the mechanisms of enzymatic stabilization of the transition state. Desolvation of the nicotinamide ring, stabilization of the oxocarbenium ion by apposition of the side chain carboxylate of Glu148 with the anomeric carbon of the ribosyl moiety, and the placement of the substrate phosphate near the positively charged side chain of His21 are all consistent with the transition state features from KIE analysis.

Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.

The cysteine protease papain is synthesized as a 40-kDa inactive precursor with a 107-amino-acid N-terminal pro region. Although sequence conservation in the pro region is lower than in the mature proteases, a conserved motif (Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp-36, papain precursor numbering) was found within the pro region of cysteine proteases of the papain superfamily. To determinate the function to this conserved motif, we have mutagenized at random each of the 4 residues individually within the pro region of the papain precursor. Precursor mutants were expressed in yeast, screened according to their ability to be processed through either a cis or trans reaction, into mature active papain. Three classes of mutants were found. Non-functional propapain mutants of the first class are completely degraded by subtilisin indicating that they are not folded into a native state. Mutants of the second class were neutral with respect to cis and trans processing. The third class included mutants that mostly accumulated as mature papain in the yeast vacuole. They had mutations that had lost the negatively charged Asp-36 residues and a mutation that probably introduces a positive charge, Phe-38His. The precursor of the Phe-38His mutant could be recovered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The Phe-38His propapain mutant has an optimum pH of autoactivation about one pH unit higher than the wild type molecule. These results indicate that the electrostatic status of the conserved motif participates in the control of intramolecular processing of the papain precursor.

An alignment/phylogeny of the papain superfamily of cysteine proteases was created using an initial structure-based alignment followed by successive iterations of sequence alignment and phylogenetic inference. The iterative approach resulted in significant improvements in the alignment/phylogeny. There were three groups of cysteine proteases that were distantly related and which could be aligned against each other only in the active site regions: the papain group, which included such stereotypical cysteine proteases as cathepsins B, C, H, L and S; and the bleomycin hydrolase and calpain groups. There was one bacterial sequence in each of the bleomycin hydrolase and calpain groups. The former probably arose by lateral gene transfer, the latter possibly by direct evolution from an ancestral protease predating the eukaryote/prokaryote divergence. The phylogeny of the papain group indicated that many families diverged almost simultaneously early during eukaryotic evolution. In mammals there are at least 12 distinct families of cysteine proteases, possibly many more, including at least two as yet uncharacterized enzymes.

Cystatin C, a cysteine protease inhibitor, was subject to hydrolysis at two sites when complexed with papain and in the presence of excess papain. A pH-dependent cleavage at His-86 increases Asp-87 was observed, as well as a pH-independent one at Gly-4 increases Lys-5. His-86 increases Asp-87 hydrolysis increased with decreasing pH and was characterized kinetically. It could be described by a single ionization with pKa = 3.4 +/- 0.2 and (kcat./Km)max. = 1.4 (+/- 0.4) x 10(4) M-1.s-1 at I = 0.3 M. C.d. spectroscopy, also at I = 0.3 M, demonstrated a conformational change with pKa = 3.2 +/- 0.2, indicating that the pH-dependence of hydrolysis was due to a conformational change in cystatin C. At I = 0.15 M, the pKa of the conformational change observed by c.d. shifted to 4.1 +/- 0.1. This indicates that at physiological ionic strength of 0.15 M, a significant proportion of cystatin C complexed with protease would be in a proteolytically labile conformation over the pH range 4.5 to 5, which is encountered in lysosomes. This may constitute a mechanism for clearing inappropriately localized cystatins. A pH-dependent conformational variability in this region of the inhibitor could explain the differences in the X-ray crystallographic and n.m.r. structures of the homologous chicken cystatin. The ionic-strength dependence of ionization indicates a hydrophobic stabilization of the ionizable group. The lack of pH-dependence of hydrolysis at Gly-4 increases Lys-5, with kcat./Km = 220 +/- 41 M-1.s-1 in the pH range 3.89 to 7.96 was unexpected in light of the normal, bell-shaped pH-dependence of papain-catalysed hydrolyses. This may reflect a different rate-limiting step of cystatin C hydrolysis.

Enzyme-substrate contacts in the hydrolysis of ester substrates by the cysteine protease papain were investigated by systematically altering backbone hydrogen-bonding and side-chain hydrophobic contacts in the substrate and determining each substrate's kinetic constants. The observed specificity energies [defined as delta delta G obs = -RT ln [(kcat/KM)first/(kcat/KM)second)]] of the substrate backbone hydrogen bonds were -2.7 kcal/mol for the P2 NH and -2.6 kcal/mol for the P1 NH when compared against substrates containing esters at those sites. The observed binding energies were -4.0 kcal/mol for the P2 Phe side chain, -1.0 kcal/mol for the P1' C=O, and -2.3 kcal/mol for the P2' NH. The latter three values probably all significantly underestimate the incremental binding energies. The P2 NH, P2 Phe side-chain, and P1 NH contacts display a strong interdependence, or cooperativity, of interaction energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the enzyme-substrate transition state. As favorable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the free energy expressed approaches the incremental interaction energy. This is the first report of a graded cooperative effect. Elucidation of favorable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.