Propagation of conformational instability in FK506-binding protein FKBP12.

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long ß4-ß5 loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The ß4-ß5 loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the ß4-ß5 loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant ß2-ß3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site.

Broad-Spectrum Small-Molecule Inhibitors Targeting the SAM-Binding Site of Flavivirus NS5 Methyltransferase.

Flavivirus infections, such as those caused by dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV), pose a rising threat to global health. There are no FDA-approved drugs for flaviviruses, although a small number of flaviviruses have vaccines. For flaviviruses or unknown viruses that may appear in the future, it is particularly desirable to identify broad-spectrum inhibitors. The NS5 protein is regarded as one of the most promising flavivirus drug targets because it is conserved across flaviviruses. In this study, we used FL-NAH, a fluorescent analog of the methyl donor S-adenosyl methionine (SAM), to develop a fluorescence polarization (FP)-based high throughput screening (HTS) assay to specifically target methyltransferase (MTase), a vital enzyme for flaviviruses that methylates the N7 and 2'-O positions of the viral 5'-RNA cap. Pilot screening identified two candidate MTase inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the DENV3 MTase with low micromolar IC50. Functional assays verified the inhibitory potency of these molecules for the flavivirus MTase activity. Binding studies indicated that these molecules are bound directly to the DENV3 MTase with similar low micromolar affinity. Furthermore, we showed that these compounds greatly reduced ZIKV replication in cell-based experiments at dosages that did not cause cytotoxicity. Finally, docking studies revealed that these molecules bind to the SAM-binding region on the DENV3 MTase, and further mutagenesis studies verified residues important for the binding of these compounds. Overall, these compounds are innovative and attractive candidates for the development of broad-spectrum inhibitors for the treatment of flavivirus infections.

A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

Isocitrate dehydrogenase 1 gene variants analysis of glioma patients from Pakistan.

Various somatic isocitrate dehydrogenase 1 (IDH1) gene variants have been reported to drive lower-grade gliomas and secondary glioblastomas. In the current study, we explored the IDH1 variants in the glioma biopsy samples of patients from Pakistan. We explored the incidence of isocitrate dehydrogenase 1 gene variants by hotspot sequencing in 80 formalin-fixed paraffin-embedded tissues of different types of glioma biopsy samples. Structural modeling of the identified variants in isocitrate dehydrogenase 1 protein was done to see their possible consequences. The frequently described p.Arg132 variants were not found in any of the glioma types. However, in our study, we identified nonsynonymous variants at the residues p.R109 and p.G136 in astrocytomas and p.R100 in oligodendroglioma. These variants are affecting a part of the conserved domain in isocitrate dehydrogenase 1. Both of p.R100 and p.R109 variants are rare and described before, whereas the p.G136 variant identified in this study has never been described previously. Structural modeling showed that variants of these residues would directly affect the substrate binding and hence the enzyme activity.

Crystal structure and transient dimerization for the FKBP12 protein from the pathogenic fungus Candida auris.

International concern over the recent emergence of Candida auris infections reflects not only its comparative ease of transmission and substantial mortality but the increasing level of resistance observed to all three major classes of antifungal drugs. Diminution in virulence has been reported for a wide range of fungal pathogens when the FK506-binding protein FKBP12 binds to that immunosuppressant drug and the binary complex then inhibits the fungal calcineurin signaling pathway. Structure-based drug design efforts have described modifications of FK506 which modestly reduce virulence for a number of fungal pathogens while also lessening the side effect of suppressing the tissue immunity response in the patient. To aid in such studies, we report the crystal structure of Candida auris FKBP12. As physiological relevance has been proposed for transient homodimerization interactions of distantly related fungal FKBP12 proteins, we report the solution NMR characterization of the homodimerization interactions of the FKBP12 proteins from both Candida auris and Candida glabrata.

Characterization of a thermostable, allosteric L-asparaginase from Anoxybacillus flavithermus.

L-asparaginase is an important enzyme with diverse applications in food industry and therapeutics. However, the enzyme currently employed in the treatment of leukaemias, comes with undesired L-glutaminase activity. A gene encoding 38 kDa L-asparaginase form Anoxybacillus flavithermus was cloned and expressed in Escherichia coli as a soluble and active enzyme. Heat treatment and Ni-affinity column chromatography provided highly purified enzyme possessing a specific activity of 165 units mg-1. The enzyme exhibited allosteric behaviour with a Hill coefficient of 1.60 and K0.5 of 25 mM with L-asparagine as specific substrate. No detectable activity was observed in the presence of D-asparagine, l-glutamine and d-glutamine. Purified AfASNase showed optimum activity at 60 °C and pH 7.0. The enzyme had ability to withstand up to 6 M urea and showed complete inactivation when treated with 1 M guanidine hydrochloride. Protein-Ligand docking and molecular dynamic simulations indicated that the regulatory site is formed by T262-T263-C265-G269-Thr294 and is located on a domain different from the one carrying the well-established active site. AfASNase is reported as first thermostable L-asparaginase with allosteric regulation. Hitherto, AfASNase presents the first characterization of recombinant L-asparaginase from the genus Anoxybacillus.

1H, 13C, 15 N chemical shift assignments of the FKBP12 protein from the pathogenic fungi Candida auris and Candida glabrata.

Multi-drug resistance is becoming an increasingly severe clinical challenge not only among pathogenic bacteria but among fungal pathogens as well. Drug design is inherently more challenging for the eukaryotic fungi due to their closer evolutionary similarity to humans. The recent rapid expansion in invasive infections throughout the world by Candida auris is of particular concern due to a substantial mortality rate, comparatively facile transmission, and an increasing level of resistance to all three of the major classes of anti-fungal drugs. One promising avenue for the development of an alternative class of anti-fungal agents currently under investigation is for drugs against the FK506-binding protein FKBP12 which, when bound to that drug, inhibits the fungal calcineurin signaling pathway with a resultant diminution in virulence. The specific challenge to this approach is that the homologous human calcineurin pathway functions in controlling the tissue immunity response, so that drug selectivity for the fungal pathway must be designed. To facilitate such efforts, we report the nearly complete backbone and sidechain resonances for the FKBP12 proteins of both Candida auris and clinically significant Candida glabrata fungi.

Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis.

Phosphoribosyl anthranilate isomerase is involved in the isomerization of phosphoribosyl anthranilate to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. In the present study, trpFGt, a gene encoding phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis, was cloned and expressed in Escherichia coli. The gene product, TrpFGt, was produced in E. coli in soluble and active form. Molecular characterization revealed that recombinant TrpFGt was highly efficient and stable. The apparent Vmax and Km values were 480 µmol min-1 mg-1 and 1.15 µM, respectively. The half-life of the enzyme was 90 min at 60 °C. Apart from thermostability, TrpFGt was highly stable against protein denaturants such as urea. There was no significant change in activity even after treatment with 8 M urea. To the best of our knowledge, TrpFGt, is the most active and stable phosphoribosyl anthranilate isomerase characterized to date and this is the first characterization of TrpF from the genus Geobacillus.

Extremely stable indole-3-glycerol-phosphate synthase from hyperthermophilic archaeon Pyrococcus furiosus.

The gene-encoding Indole-3-glycerol phosphate synthase, a key enzyme involved in the cyclization of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate, from Pyrococcus furiosus was cloned and expressed in Escherichia coli. The gene product was produced in the soluble and active form. The recombinant protein, purified to apparent homogeneity, displayed highest activity at 100 °C and pH of 5.5. The recombinant enzyme followed Michaelis-Menten kinetics exhibiting apparent Vmax and Km values of 20 ± 0.5 µmol min-1 mg-1 and 140 ± 10 µM, respectively. The activation energy, determined from the linear Arrhenius plot, was 17 ± 0.5 kJ mol-1. A unique property of PfInGPS is its stability against denaturants and temperature. There was no significant change in activity even in the presence of 8 M urea or 5 M guanidine hydrochloride. Furthermore, recombinant PfInGPS was highly thermostable with a half-life of 200 min at 100 °C. To the best of our knowledge, this is the most stable indole-3-glycerol phosphate synthase characterized to date.

Pcal_0111, a highly thermostable bifunctional fructose-1,6-bisphosphate aldolase/phosphatase from Pyrobaculum calidifontis.

Pyrobaculum calidifontis genome harbors an open reading frame Pcal_0111 annotated as fructose bisphosphate aldolase. Although the gene is annotated as fructose bisphosphate aldolase, it exhibits a high homology with previously reported fructose-1,6-bisphosphate aldolase/phosphatase from Thermoproteus neutrophilus. To examine the biochemical properties of Pcal_0111, we have cloned and expressed the gene in Escherichia coli. Purified recombinant Pcal_0111 catalyzed both phosphatase and aldolase reactions with specific activity values of 4 U and 1.3 U, respectively. These values are highest among the fructose 1,6-bisphosphatases/aldolases characterized from archaea. The enzyme activity increased linearly with the increase in temperature until 100 °C. Recombinant Pcal_0111 is highly stable with a half-life of 120 min at 100 °C. There was no significant change in the circular dichroism spectra of the protein up to 90 °C. The enzyme activity was not affected by AMP but strongly inhibited by ATP with an IC50 value of 0.75 mM and mildly by ADP. High thermostability and inhibition by ATP make Pcal_0111 a unique fructose 1,6-bisphosphatase/aldolase.

Genetic variants in FGFR2 and TNRC9 genes are associated with breast cancer risk in Pakistani women.

Single nucleotide polymorphisms (SNPs) lead to genetic differences in breast cancer (BC) susceptibility among women from different ethnicities. The present study aimed at investigating the involvement of SNPs of three genes, including fibroblast growth factor receptor 2 (FGFR2), trinucleotide-repeat-containing 9 (TNRC9) and mitogen-activated protein kinase kinase kinase 1 (MAP3K1), as risk factors for the development of BC. A case­control study (90­100 cases; 90­100 controls) was performed to evaluate five genetic variants of three genes, including FGFR2 (SNPs: rs1219648, rs2981582), TNRC9 (SNPs: rs8051542, rs3803662) and MAP3K1 (SNP: rs889312) as BC risk factors in Pakistani women. Significant associations were observed between BC risk and two SNPs of FGFR2 [rs2981582 (P=0.005), rs1219648 (P=9.08e­006)] and one SNP of TNRC9 [rs3803662) (P=0.012)] in Pakistani women. On examining the different interactions of these SNPs with various clinicopathological characteristics, all three associated genetic variants, rs2981582 rs1219648 and rs3803662, exhibited a greater predisposition to sporadic, in comparison to familial, BC. Furthermore, there was an increased effect of BC risk between haplotype combinations of the two SNPs of FGFR2 (rs2981582 and rs1219648) in Pakistani women. The results of the present study suggest that variants of FGFR2 and TNRC9 may contribute to the genetic susceptibility of BC in Pakistani women.

Pcal_1127, a highly stable and efficient ribose-5-phosphate pyrophosphokinase from Pyrobaculum calidifontis.

Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM-1 s-1. These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.

Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.

Engineering specificity in a dynamic protein complex with a single conserved mutation.

It has been demonstrated that the complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP) exists as a delicate equilibrium of a specific, active state and the non-specific, dynamic encounter state. An ortholog of yeast Cc, horse Cc, binds CcP but forms a much more dynamic complex, as demonstrated by NMR spectroscopy. A single conservative mutation of lysine 13 to arginine reduces the dynamics and enhances the specificity. The crystal structure of the stereospecific complex resembles the yeast Cc-CcP complex. In contrast, the K13A mutation increases the dynamic nature of the complex with CcP, showing that specificity in a redox protein complex can depend on the interactions of a single side chain in the binding interface.

TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH.

Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus kodakaraensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized. Molecular weight of the recombinant protein was 49,375 Da when determined by matrix-assisted laser desorption/ionization time-of-flight and 300 kDa when analyzed by gel filtration chromatography indicating that it existed in a hexameric form. The enzyme was highly thermostable even in boiling water where it exhibited more than 95% of the enzyme activity after incubation of 150 min. TK1299 catalyzed the oxidation of NADH as well as NADPH and predominantly converted O2 to H2O (more than 75%). K(m) value of the enzyme towards NADH and NADPH was almost same (24 ± 2 µM) where as specific activity was higher with NADPH compared to NADH. To our knowledge this is the most thermostable and unique NAD(P)H oxidase displaying higher enzyme activity with NADPH.

Communication between (L)-galactono-1,4-lactone dehydrogenase and cytochrome c.

UNLABELLED: l-galactono-1,4-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plant mitochondria. Here we investigated the communication between Arabidopsis thaliana GALDH and its natural electron acceptor cytochrome c (Cc). Using laser-generated radicals we observed the formation and stabilization of the GALDH semiquinone anionic species (GALDHSQ ). GALDHSQ oxidation by Cc exhibited a nonlinear dependence on Cc concentration consistent with a kinetic mechanism involving protein-partner association to form a transient bimolecular complex prior to the electron transfer step. Oxidation of GALDHSQ by Cc was significantly impaired at high ionic strength, revealing the existence of attractive charge-charge interactions between the two reactants. Isothermal titration calorimetry showed that GALDH weakly interacts with both oxidized and reduced Cc. Chemical shift perturbations for (1) H and (15) N nuclei of Cc, arising from the interactions with unlabeled GALDH, were used to map the interacting surface of Cc. For Arabidopsis Cc and yeast Cc, similar residues are involved in the interaction with GALDH. These residues are confined to a single surface surrounding the heme edge. The range of chemical shift perturbations for the physiological Arabidopsis Cc-GALDH complex is larger than that of the non-physiological yeast Cc-GALDH complex, indicating that the former complex is more specific. In summary, the results point to a relatively low affinity GALDH-Cc interaction, similar for all partner redox states, involving protein-protein dynamic motions. Evidence is also provided that Cc utilizes a conserved surface surrounding the heme edge for the interaction with GALDH and other redox partners. DATABASE: NMR assignment of the backbone amide resonances of Arabidopsis CcRED has been deposited in BMRB database (BMRB accession number 18828). L-galactono-1,4-lactone dehydrogenase (L-galactono-1,4-lactone: ferricytochrome c oxidoreductase, EC 1.3.2.3).

Electrochemical determination of hydrogen peroxide with cytochrome c peroxidase and horse heart cytochrome c entrapped in a gelatin hydrogel.

A novel and versatile method, based on a membrane-free enzyme electrode in which both the enzyme and a mediator protein are entrapped in a gelatine hydrogel was developed for the fabrication of biosensors. As a proof of principle, we prepared a hydrogen peroxide biosensor by successfully entrapping both horse heart cytochrome c (HHC) and Saccharomyces cerevisae cytochrome c peroxidase (CCP) in a gelatin matrix which is immobilized on a gold electrode. This electrode was first pretreated with 6-mercaptohexanol. The biosensor displayed a rapid response and an expanded linear response range from 0 to 0.3 mM (R = 0.987) with a detection limit of 1 × 10(-5)M in a HEPES buffer solution (pH 7.0). This method of encapsulation is now further investigated for industrial biosensor applications.

Dynamics in electron transfer protein complexes.

Electron transfer proteins transport electrons safely between large redox enzymes. The complexes formed by these proteins are among the most transient. The biological function requires, on the one hand, sufficient specificity of the interaction to allow for rapid and selective electron transfer, and, on the other hand, a fast turnover of the complex. Recent progress in the characterization of the nature of these complexes has demonstrated that the encounter state plays an important role. This state of initial binding is dominated by electrostatic interactions, and consists of an ensemble of orientations. Paramagnetic relaxation enhancement NMR and chemical shift perturbation analysis provide ways for the experimental characterisation of the encounter state. Several studies that have used these techniques have shown that the surface area sample in the encounter state can be limited to the immediate environment of the final, specific complex. The encounter complex can represent a large fraction and, in some small complexes, no specific binding is detected at all. It can be concluded that, in electron transfer protein complexes, a fine balance is sought between the low-specificity encounter state and the high-specificity productive complex to meet the opposing requirements of rapid electron transfer and a high turnover rate.

Shifting the equilibrium between the encounter state and the specific form of a protein complex by interfacial point mutations.

Recent experimental studies have confirmed a long-held view that protein complex formation proceeds via a short-lived encounter state. The population of this transient intermediate, stabilized mainly by long-range electrostatic interactions, varies among different complexes. Here we show that the occupancy of the encounter state can be modulated across a broad range by single point mutations of interfacial residues. Using a combination of Monte Carlo simulations and paramagnetic relaxation enhancement NMR spectroscopy, we illustrate that it is possible to both enhance and diminish the binding specificity in an electron transfer complex of yeast cytochrome c (Cc) and cytochrome c peroxidase. The Cc T12A mutation decreases the population of the encounter to 10% as compared with 30% in the wild-type complex. More dramatically, the Cc R13A substitution reverses the relative occupancies of the stereospecific and the encounter forms, with the latter now being the dominant species with the population of 80%. This finding indicates that the encounter state can make a large contribution to the stability of a protein complex. Also, it appears that by adjusting the amount of the encounter through a judicious choice of point mutations, we can remodel the energy landscape of a protein complex and tune its binding specificity.

Mechanistic and stereochemical studies of glycine oxidase from Bacillus subtilis strain R5.

Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein M(r) of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M(r) of 785.1 (theoretical value of 785.5). Glycine oxidase optimally catalyzes the conversion of glycine and oxygen into glyoxylate, hydrogen peroxide, and ammonia. Using samples of [2-RS-(3)H(2),2-(14)C]-, [2-R-(3)H,2-(14)C]-, and [2-S-(3)H,2-(14)C]glycine, we found that in the overall process H(Si) is removed. Incubation of the enzyme with [2-RS-(3)H(2),2-(14)C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, led to the recovery of 98.5% of the original glycine, which had the same (3)H:(14)C ratio as the starting substrate. The primary isotope effect was studied using [2-(2)H(2)]glycine, and we found that the specificity constants, k(cat)/K(M), for the protio and deuterio substrates were 1.46 x 10(3) and 1.05 x 10(2) M(-1) s(-1), respectively. Two alternative mechanisms for FAD-containing oxidases that involve either the intermediacy of a FADH(2)-imino acid complex or an amino acid covalently linked to FAD, formed via a carbanion, have been considered. The current knowledge of the mechanisms is reviewed, and we argue that a mechanism involving the FADH(2)-imino acid complex can be dissected to satisfactorily explain some of puzzling observations for which the carbanion mechanism was originally conceived. Furthermore, our results, together with observations in the literature, suggest that the interaction of glycine with the enzyme occurs within a tight ternary complex, which is protected from the protons of the medium.