Active Site Titration Research Articles

Development of an active-site titrant for SARS-CoV-2 main protease as an indispensable tool for evaluating enzyme kinetics

A titrant for the SARS-CoV-2 main protease (Mpro) was developed that enables, for the first time, the exact determination of the concentration of the enzymatically active Mpro by active-site titration. The covalent binding mode of the tetrapeptidic titrant was elucidated by the determination of the crystal structure of the enzyme–titrant complex. Four fluorogenic substrates of Mpro, including a prototypical, internally quenched Dabcyl-EDANS peptide, were compared in terms of solubility under typical assay conditions. By exploiting the new titrant, key kinetic parameters for the Mpro-catalyzed cleavage of these substrates were determined.

PENICILLIN ACYLASE: A RETROSPECTIVE OF STUDYING THE KINETICS AND THERMODYNAMICS OF PRACTICALLY SIGNIFICANT REACTIONS

The review considers the contribution of works carried out at the scientific school of Ilya Vasilievich Berezin to the study of the kinetics and thermodynamics of penicillin acylase-catalyzed reactions. Methods for determining the activity of penicillin acylases, the reversibility of the enzymatic hydrolysis of a number of penicillins, cephalosporins and related compounds, the influence of the β-lactam ring on the thermodynamics of the synthesis of new penicillins and cephalosporins by direct condensation as well as by acyl transfer, the issues of optimizing the conditions for enzymatic acyl transfer and the use of supersaturated reagent solutions are discussed. The role of chromogenic substrates in the study of penicillin acylase, the possibility of using the methods of titration of active sites of the enzyme and the creation of “smart” biocatalysts based on penicillin acylase due to the formation of conjugates with stimulus-sensitive polymers are considered.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

Coulometric Titration of Active Sites at Mesostructured Cobalt Oxide Spinel by Surface Interrogation Mode of Scanning Electrochemical Microscopy

Cobalt-based transition-metal oxides are promising candidates for the oxygen evolution reaction (OER). However, a complex interplay between the catalyst crystal structures and material morphologies...

Kringles of substrate plasminogen provide a 'catalytic switch' in plasminogen to plasmin turnover by Streptokinase.

To understand the role of substrate plasminogen kringles in its differential catalytic processing by the streptokinase - human plasmin (SK-HPN) activator enzyme, Fluorescence Resonance Energy Transfer (FRET) model was generated between the donor labeled activator enzyme and the acceptor labeled substrate plasminogen (for both kringle rich Lys plasminogen - LysPG, and kringle less microplasminogen - µPG as substrates). Different steps of plasminogen to plasmin catalysis i.e. substrate plasminogen docking to scissile peptide bond cleavage, chemical transformation into proteolytically active product, and the decoupling of the nascent product from the SK-HPN activator enzyme were segregated selectively using (1) FRET signal as a proximity sensor to score the interactions between the substrate and the activator during the cycle of catalysis, (2) active site titration studies and (3) kinetics of peptide bond cleavage in the substrate. Remarkably, active site titration studies and the kinetics of peptide bond cleavage have shown that post docking chemical transformation of the substrate into the product is independent of kringles adjacent to the catalytic domain (CD). Stopped-flow based rapid mixing experiments for kringle rich and kringle less substrate plasminogen derivatives under substrate saturating and single cycle turnover conditions have shown that the presence of kringle domains adjacent to the CD in the macromolecular substrate contributes by selectively speeding up the final step, namely the product release/expulsion step of catalysis by the streptokinase-plasmin(ogen) activator enzyme.

Methanol oxidative dehydrogenation and dehydration on carbon nanotubes: active sites and basic reaction kinetics

In situ active site titration, carbonyl group containing model catalysts, and kinetic analysis have been applied to reveal the nature of oxidized carbon nanotubes catalyzed methanol dehydration and oxidative dehydrogenation reactions.

Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress

BackgroundFully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of H2O2 in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14. MethodsThe proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined. ResultsUnder osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a KD of ~25 μM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular H2O2 scavenging enzyme, with a KD of ~0.13 μM, and protects it from dehydration-induced loss of activity. ConclusionsWe propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions. General significanceERD14 has a direct effect on the activity of redox enzymes.

Oxidative dehydrogenation on nanocarbon: Effect of heteroatom doping

Kinetic analysis is a powerful and effective strategy to reveal the physical-chemical nature of the promotion effect of heteroatoms to nanocarbon catalysts. The present work reported the mechanistic and kinetic analysis of ethylbenzene oxidative dehydrogenation (ODH) reactions on heteroatom (N or B) doped and undoped carbon nanotube (CNT) catalysts via active site titration, kinetic isotope effect and single reactant surface reaction experiments etc. The physical-chemical meanings behind the elementary step rate and equilibrium constants were revealed and applied for interpretations of the promotion effect of heteroatoms at molecular level. Nitrogen doped CNT exhibited both higher rate and equilibrium constants for C–H bond dissociation and O2 adsorption than undoped one via facilitating the electron transfer process. The evolution of the active sites could be quantitatively described with rate equation via the theory of most abundant surface intermediates, which provides in depth understandings on the mechanism and structure-function relations in carbon catalyzed redox reactions.

Methods for determining the intrinsic kinetic characteristics of irreversible adsorption processes

To benchmark the performance of a different of catalytic materials it is essential to know the adsorption characteristics such as the kinetic order of adsorption, the apparent rate constant and the total number of active sites. It is advantageous to normalize the reaction rate to the number of active sites; a quantity commonly referred to as the turnover frequency (TOF). Methods such as chemisorption and SSITKA (steady state isotopic transient kinetic analysis) are conventionally used to quantify the number of active sites, both have advantages and drawbacks. The TAP (Temporal Analysis of Products) pulse response technique provides a distinct method for precise (10 nmol) quantification of active sites at elevated temperatures. Using irreversible reaction processes, mathematical techniques for analysis of pulsed active site titration are described herein. Whereas previous methods simply consider the total capacity for gas adsorption, these new methods take into account the dynamics and interdependence of the conversion, the number of available sites and the adsorption rate as they change over the course of the titration experiment. Moreover, these methods enable an independent fitting of the kinetic order and stoichiometry of the adsorption process directly from experimental data. In comparison with experimental data for the incremental oxidation of a reduced platinum, we found the analytical method of linear rectification was most robust and efficient. In fitting the kinetic order to experimental data collected between 125 and 175 °C we found a clear indication over the range of 45–95% conversion of a second order rate dependence and 2:1 balance between active sites and O2.

Oxidative dehydrogenation of ethylbenzene on nanocarbon: Kinetics and reaction mechanism

Active site titration, kinetic analysis, isotope and temperature programmed surface reaction experiments have been applied to reveal the nature of nanocarbon catalyzed alkane oxidative dehydrogenation reactions. The catalytic reaction involves the redox cycle of ketonic carbonyl-hydroxyl pairs on nanocarbon materials, during which the substrate of ethylbenzene (EB) dehydrogenates to product styrene, and the molecular oxygen re-oxidizes the catalysts yielding water. The rate equation based on the mechanistic interpretations of the catalytic reactions provides intrinsic rate or equilibrium constants, which have their specific physicochemical meanings and could be directly linked to the catalytic process. The proposed rate equation could also accurately describe the effects of reactant (EB or O2) pressures and isotopomers (deuterated-EB or 18O2) on rate data, and provide a quantitative analysis on the evolution and content of surface intermediates on carbon catalysts, which is a reliable descriptor of the intrinsic reactivity and essential to establish the structure-function relations for carbon catalyzed redox reactions.

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples.

A novel methodology for in situ redox active site titration of TiO2-supported vanadia during ethanol partial oxidation catalysis

An advanced titration method during ethanol partial oxidation with tert-butanol is proposed to quantify the number of active redox sites in supported vanadia-titania catalytic systems (VOx/TiO2). In agreement with previous reports, our results indicate that not all vanadia atoms equally participate in the catalytic redox cycle. Therefore, the use of total vanadium loadings to calculate turnover frequencies results in an underestimation of actual turnover frequency values. The kinetically-relevant rate constants for ethanol partial oxidation obtained on the basis on the number of active sites calculated by our method indicate that these rate constants are independent of vanadia loading. This in turn suggests that the main species active for ethanol partial oxidation are well dispersed small sized vanadia clusters with a small number of VOV bridging bonds, predominant at low vanadia coverages.

Ultrasensitive Fluorogenic Reagents for Neuraminidase Titration.

Influenza viral neuraminidase plays a crucial role during infections. It is a major target for the development of anti-influenza drugs and is also attracting increasing attention as a vaccine target as evidence accumulates that neuraminidase-neutralizing antibodies contribute to protection. However, no method currently exists to accurately and efficiently measure concentrations of active neuraminidase in virus samples or other crude mixtures, which hampers development on both fronts. In this report, we describe the development of a selective and sensitive active-site titration reagent for neuraminidase that can quantify viral neuraminidases down to sub-nanomolar levels in crude samples, with no background from non-viral neuraminidases. By using this reagent, we determined accurate kcat values for six influenza A and two influenza B neuraminidases for the first time. We also quantified the neuraminidase content in a commercial influenza vaccine, thus demonstrating that this titration reagent opens the possibility for better vaccine analysis.

Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia.

LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). The dissociation constant (Kd ) for activated platelet binding was in the 1 μm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat /Km values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s-1 /0.32 μm and 3.3 s-1 /0.44 μm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103 m-1 s-1 . The Kd values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kd for binding to soluble EPCR was 41 nm. Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.

Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV

Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO2 hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs.

An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures.

Urzymes-short, active core modules derived from enzyme superfamilies-prepared from the two aminoacyl-tRNA synthetase (aaRS) classes contain only the modules shared by all related family members. They have been described as models for ancestral forms. Understanding them currently depends on inferences drawn from the crystal structures of the full-length enzymes. As aaRS Urzymes lack much of the mass of modern aaRS's, retaining only a small portion of the hydrophobic cores of the full-length enzymes, it is desirable to characterize their structures. We report preliminary characterization of (15)N tryptophanyl-tRNA synthetase Urzyme by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. The limited dispersion of (1)H chemical shifts (0.5 ppm) is inconsistent with a narrow ensemble of well-packed structures in either free or substrate-bound forms, although the number of resonances from the bound state increases, indicating a modest, ligand-dependent gain in structure. Circular dichroism spectroscopy shows the presence of helices and evidence of cold denaturation, and all ligation states induce Sypro Orange fluorescence at ambient temperatures. Although the term "molten globule" is difficult to define precisely, these characteristics are consistent with most such definitions. Active-site titration shows that a majority of molecules retain ∼60% of the transition state stabilization free energy observed in modern synthetases. In contrast to the conventional view that enzymes require stable tertiary structures, we conclude that a highly flexible ground-state ensemble can nevertheless bind tightly to the transition state for amino acid activation.

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

A novel form of ficin from Ficus carica latex: Purification and characterization

A novel ficin form, named ficin E, was purified from fig tree latex by a combination of cation-exchange chromatography on SP-Sepharose Fast Flow, Thiopropyl Sepharose 4B and fplc-gel filtration chromatography. The new ficin appeared not to be sensitive to thiol derivatization by a polyethylene glycol derivative, allowing its purification. The protease is homogeneous according to PAGE, SDS–PAGE, mass spectrometry, N-terminal micro-sequencing analyses and E-64 active site titration. N-terminal sequencing of the first ten residues has shown high identity with the other known ficin (iso)forms. The molecular weight was found to be (24,294±10)Da by mass spectrometry, a lower value than the apparent molecular weight observed on SDS–PAGE, around 27kDa. Far-UV CD data revealed a secondary structure content of 22% α-helix and 26% β-sheet. The protein is not glycosylated as shown by carbohydrate analysis. pH and temperature measurements indicated maxima activity at pH 6.0 and 50°C, respectively. Preliminary pH stability analyses have shown that the protease conserved its compact structure in slightly acidic, neutral and alkaline media but at acidic pH (<3), the formation of some relaxed or molten state was evidenced by 8-anilino-1-naphtalenesulfonic acid binding characteristics. Comparison with the known ficins A, B, C, D1 and D2 (iso)forms revealed that ficin E showed activity profile that looked like ficin A against two chromogenic substrates while it resembled ficins D1 and D2 against three fluorogenic substrates. Enzymatic activity of ficin E was not affected by Mg2+, Ca2+ and Mn2+ at a concentration up to 10mM. However, the activity was completely suppressed by Zn2+ at a concentration of 1mM. Inhibitory activity measurements clearly identified the enzyme as a cysteine protease, being unaffected by synthetic (Pefabloc SC, benzamidine) and by natural proteinaceous (aprotinin) serine proteases inhibitors, by aspartic proteases inhibitors (pepstatin A) and by metallo-proteases inhibitors (EDTA, EGTA). Surprisingly, it was well affected by the metallo-protease inhibitor o-phenanthroline. The enzymatic activity was however completely blocked by cysteine proteases inhibitors (E-64, iodoacetamide), by thiol-blocking compounds (HgCl2) and by cysteine/serine proteases inhibitors (TLCK and TPCK). This is a novel ficin form according to peptide mass fingerprint analysis, specific amidase activity, SDS–PAGE and PAGE electrophoretic mobility, N-terminal sequencing and unproneness to thiol pegylation.

Active site titration of immobilized beta-galactosidase for the determination of active enzymes

In the present study, an active site titration method is demonstrated, to determine the amount of active enzyme (β-galactosidase), immobilized on a support. Two types of supports were investigated, viz. amino acrylic resin and a mixed matrix membrane. Furthermore, 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-d-galactopyranoside was used as an inhibitor for the active site titration of immobilized β-galactosidase obtained from Kluyveromyces lactis. Using the active site titration, approximately 8.3mg of active enzyme was found on 1g of dried commercially available SPRIN imibond, which is an amino acrylic resin with covalently bound β-galactosidase obtained from K. lactis. However, this method, in its present form, was not effective on the mixed matrix membranes due to the irreversible partial adsorption of the leaving group (2′,4′-dinitrophenolate) by the membrane. This observation implied that it is important to investigate interactions between the support and the used inhibitor and leaving group.

Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris

Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man)5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDS–PAGE as a single band of 30kDa and treatment with peptide N-glycosidase F decreased this to 25kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced KM, whereas heparin effects on kcat were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns.