pH-rate Profile Research Articles

Exploring the catalytic mechanism of the 10-23 DNAzyme: insights from pH-rate profiles.

The 10-23 DNAzyme, a catalytic DNA molecule with RNA-cleaving activity, has garnered significant interest for its potential therapeutic applications as a gene-silencing agent. However, the lack of a detailed understanding about its mechanism has hampered progress. A recent structural analysis has revealed a highly organized conformation thanks to the stabilization of specific interactions within the catalytic core of the 10-23 DNAzyme, which facilitate the cleavage of RNA. In this configuration, it has been shown that G14 is in good proximity to the cleavage site which suggests its role as a general base, by activating the 2'-OH nucleophile, in the catalysis of the 10-23 DNAzyme. Also, the possibility of a hydrated metal acting as a general acid has been proposed. In this study, through activity assays, we offer evidence of the involvement of general acid-base catalysis in the mechanism of the 10-23 DNAzyme by analyzing its pH-rate profiles and the role of G14, and metal cofactors like Mg2+ and Pb2+. By substituting G14 with its analogue 2-aminopurine and examining the resultant pH-rate profiles, we propose the participation of G14 in a catalytically relevant proton transfer event, acting as a general base. Further analysis, using Pb2+ as a cofactor, suggests the capability of the hydrated metal ion to act as a general acid. These functional results provide critical insights into the catalytic strategies of RNA-cleaving DNAzymes, revealing common mechanisms among nucleic acid enzymes that cleave RNA.

SHP-1 Variants Broaden the Understanding of pH-Dependent Activities in Protein Tyrosine Phosphatases.

The protein tyrosine phosphatase (PTP) SHP-1 plays an important role in both immune regulation and oncogenesis. This enzyme is part of a broader family of PTPs that all play important regulatory roles in vivo. Common to these enzymes is a highly conserved aspartic acid (D421 in SHP-1) that acts as an acid/base catalyst during the PTP-catalyzed reaction. This residue is located on a mobile loop, the WPD-loop, the dynamic behavior of which is intimately connected to the catalytic activity. The SHP-1 WPD-loop variants H422Q, E427A, and S418A have been kinetically characterized and compared to those of the wild-type (WT) enzyme. These variants exhibit limiting magnitudes of k cat ranging from 43 to 77% of the WT enzyme. However, their pH profiles are significantly broadened in the basic pH range. As a result, above pH 6, the E427A and S418A variants have turnover numbers notably higher than those of WT SHP-1. Molecular modeling results indicate that the shifted pH dependencies result primarily from changes in solvation and hydrogen-bonding networks that affect the pK a of the D421 residue, explaining the changes in pH-rate profiles for k cat on the basic side. In contrast, a previous study of a noncatalytic residue variant of the PTP YopH, which also exhibited changes in pH dependency, showed that the catalytic change arose from mutation-induced changes in conformational equilibria of the WPD-loop. This finding and the present study show the existence of distinct strategies for nature to tune the activity of PTPs in particular environments through controlling the pH dependency of catalysis.

Solubility, pH-Solubility Profile, pH-Rate Profile, and Kinetic Stability of the Tyrosine Kinase Inhibitor, Alectinib.

Alectinib HCl (ALBHCl) is a tyrosine kinase inhibitor used for non-small cell lung carcinoma (NSCLC). The aim of this study is to unlock some of the physicochemical properties of ALBHCL that serve as a database for any future studies. A solubility study of ALBHCL was performed in different solvents. Also, photostability was tested in the solution and solid states, and the order of reaction and rate constant were calculated. In addition to the pH solubility relation, the pH-rate relation at different temperatures was also studied, and the profiles were constructed. A solubility study was also performed in different media for the purpose of optimizing suitable sink conditions for the in vitro dissolution testing of solid dosage forms. Solubility tests in multiple solvents and pH conditions revealed that the highest solubility was in DMSO, methanol, and chloroform, with acidic media yielding the maximum solubility but degrading at rather low pH levels. ALBHCL proved unstable at high temperatures and under light exposure, with varying stability across different pH levels. The optimal dissolution media for in vitro oral dosage form evaluation were determined, achieving sink conditions at pH levels of 6.8 and 4.5 with specific additives. This study enhances the existing database on ALBHCL's physicochemical properties, emphasizing the importance of pH optimization in pharmaceutical processes and providing valuable insights into its pharmaceutical application.

Snapshots of the Reaction Coordinate of a Thermophilic 2'-Deoxyribonucleoside/ribonucleoside Transferase.

Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2'-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2'-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2'-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2'-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5-9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2'-fluoro-2'-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2'-difluoro-2'-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2'-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.

Photolysis of tolfenamic acid in aqueous and organic solvents: a kinetic study.

Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug that was studied for its photodegradation in aqueous (pH 2.0-12.0) and organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol). TA follows first-order kinetics for its photodegradation, and the apparent first-order rate constants (k obs) are in the range of 0.65 (pH 12.0) to 6.94 × 10-2 (pH 3.0) min-1 in aqueous solution and 3.28 (1-butanol) to 7.69 × 10-4 (acetonitrile) min-1 in organic solvents. The rate-pH profile for TA photodegradation is an inverted V (∧) or V-top shape, indicating that the cationic form is more susceptible to acid hydrolysis than the anionic form of TA, which is less susceptible to alkaline hydrolysis. The fluorescence behavior of TA also exhibits a V-top-shaped curve, indicating maximum fluorescence intensity at pH 3.0. TA is highly stable at a pH range of 5.0-7.0, making it suitable for formulation development. In organic solvents, the photodegradation rate of TA increases with the solvent's dielectric constant and solvent acceptor number, indicating solute-solvent interactions. The values of k obs decreased with increased viscosity of the solvents due to diffusion-controlled processes. The correlation between k obs versus ionization potential and solvent density has also been established. A total of 17 photoproducts have been identified through LC-MS, of which nine have been reported for the first time. It has been confirmed through electron spin resonance (ESR) spectrometry that the excited singlet state of TA is converted into an excited triplet state through intersystem crossing, which results in an increased rate of photodegradation in acetonitrile.

Crystal Structure, Steady-State, and Pre-Steady-State Kinetics of Acinetobacter baumannii ATP Phosphoribosyltransferase.

The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisGS, is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in kcat. Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisGS) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, kcat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisGS. The HisGS-catalyzed reaction is limited by the chemical step, as suggested by the enhancement of kcat when Mg2+ was replaced by Mn2+, and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than kcat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.

Neutral Phenolic Contaminants Are Not Necessarily More Resistant to Permanganate Oxidation Than Their Dissociated Counterparts: Importance of Proton-Coupled Electron Transfer.

Despite decades of research on phenols oxidation by permanganate, there are still considerable uncertainties regarding the mechanisms accounting for the unexpected parabolic pH-dependent oxidation rate. Herein, the pH effect on phenols oxidation was reinvestigated experimentally and theoretically by highlighting the previously unappreciated proton transfer. The results revealed that the oxidation of protonated phenols occurred via proton-coupled electron transfer (PCET) pathways, which can switch from ETPT (electron transfer followed by proton transfer) to CEPT (concerted electron-proton transfer) or PTET (proton transfer followed by electron transfer) with an increase in pH. A PCET-based model was thus established, and it could fit the kinetic data of phenols oxidation by permanganate well. In contrast with what was previously thought, both the simulating results and the density functional theory calculation indicated the rate of CEPT reaction of protonated phenols with OH- as the proton acceptor was much higher than that of deprotonated phenols, which could account for the pH-rate profiles for phenols oxidation. Analysis of the quantitative structure-activity relationships among the modeled rate constants, Hammett constants, and pKa values of phenols further supports the idea that the oxidation of protonated phenols is dominated by PCET. This study improves our understanding of permanganate oxidation and suggests a new pattern of reactivity that may be applicable to other systems.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.

The enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid–base catalysis and substrate-dependent metal ion activation

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.

A Systematic Degradation Kinetics Study of Dalbavancin Hydrochloride Injection Solutions

The degradation kinetics of the glycopeptide antibiotic dalbavancin in solution are systematically evaluated over the pH range 1–12 at 70°C. The decomposition rate of dalbavancin was measured as a function of pH, buffer composition, temperature, ionic strength, and drug concentration. A pH-rate profile was constructed using pseudo first-order kinetics at 70°C after correcting for buffer effects; the observed pH-rate profile could be fitted with standard pseudo first order rate laws. The degradation reactions of dalbavancin were found to be strongly dependent on pH and were catalyzed by protons or hydroxyl groups at extreme pH values. Dalbavancin shows maximum stability in the pH region 4–5. Based on the Arrhenius equation, dalbavancin solution at pH 4.5 is predicted to have a maximum stability of thirteen years under refrigerated conditions, eight months at room temperature and one month at 40°C. Mannosyl Aglycone (MAG), the major thermal and acid degradation product, and DB-R6, an additional acid degradation product, were formed in dalbavancin solutions at 70°C due to hydrolytic cleavage at the anomeric carbons of the sugars. Through deamination and hydrolytic cleavage of dalbavancin, a small amount of DB-Iso-DP2 (RRT-1.22) degradation product was also formed under thermal stress at 70°C. A greater amount of the base degradation product DB-R2 forms under basic conditions at 70°C due to epimerization of the alpha carbon of phenylglycine residue 3.

Altered Mechanisms for Acid-Catalyzed RNA Cleavage and Isomerization Reactions Models.

RNA strand cleavage by 2'-O-transphosphorylation is catalyzed not only by numerous nucleolytic RNA enzymes (ribozymes) but also by hydroxide or hydronium ions. In experiments, both cleavage of the 5'-linked nucleoside and isomerization between 3',5'- and 2',5'-phosphodiesters occur under acidic conditions, while only the cleavage reaction is observed under basic conditions. An ab initio path-integral approach for simulating kinetic isotope effects is used to reveal the reaction mechanisms for RNA cleavage and isomerization reactions under acidic conditions. Moreover, the proposed mechanisms can also be combined through the experimental pH-rate profiles.

Hydrolysis kinetics of the prodrug myristyl nicotinate

Myristyl nicotinate is a prodrug of nicotinic acid. In this research, the kinetics of hydrolysis for myristyl nicotinate was studied in an aqueous phosphate buffer solution within a 5–10 pH range and constant ionic strength at a high temperature which was 80 °C to perform accelerated hydrolysis experiments. The effect of temperature, ionic strength, buffer concentrations, and buffer type was studied. The degradation was monitored using a validated HPLC method. The kinetics of hydrolysis of myristyl nicotinate was also studied in skin and liver homogenates. The hydrolysis was found to follow pseudo-first-order kinetics. The rate constant was calculated from the slope of a linear plot of Ln transformation (Ln) of the remaining parent prodrug concentration versus time. The hydrolysis was found pH- dependent, and a pH rate profile was constructed. Moreover, the hydrolysis rate of the prodrug was found to be buffer species dependent. Carbonate buffer has the most catalytic effect over borate and phosphate buffers. The effect of temperature on the kinetics of hydrolysis of myristyl nicotinate in phosphate buffer at pH 9 at 343, 348, 353, and 358°K was studied. The hydrolysis was found to follow the Arrhenius equation. From the Arrhenius plot, the half-life at 25 °C, and the activation energy were calculated and were found to be 466.5 days and 24.57 kcal mol−1, respectively. The hydrolysis of the prodrug was faster in liver and skin homogenates than those in aqueous buffer solutions. The pseudo-first-order rate constants were found to be 0.012, 0.028 min−1 for myristyl nicotinate in the liver, and skin homogenates, respectively.

Mechanistic studies on the oxidation reaction of antitubercular drug isoniazid and its analogy hydrazides by chlorine dioxide over a wide pH range

Oxidations of the antitubercular drug isoniazid (INH) and its analogy hydrazides, nicotinic hydrazide (NH), picolinoyl hydrazide (PH), and benzohydrazide (BH), by ClO2 were investigated with a focus on the kinetics and reaction mechanisms. In buffered solutions of a wide pH range (from 0.51 to 10.35), the oxidation reactions strictly followed the second-order kinetics, and the apparent second-order rate constant kapp versus pH profiles were established at 25.0 °C and 1.0 M ionic strength. Excess ClO2 could rapidly oxidize the hydrazides to their corresponding aryl acids which were identified by mass spectrometry. For each of the hydrazides, the proposed reaction mechanism involves parallel rate-determining reactions between ClO2 and various protolysis species of the hydrazide, rendering two types of hydrazyl free radicals; the free radicals are quickly oxidized by 3 more equivalents of ClO2 to aryl acids. The derived rate constants for the rate-determining reactions revealed that for each hydrazide a huge reactivity difference existed among the protolysis species (>108 times) and that the enolate form (or its resonant structures) was extremely reactive. Two possible modes of electron transfer in the rate-determining reactions are discussed based on the observed reaction characters. In buffered solution of pH 7.0 and 25.0 °C, the measured values of kapp are 6.20×104, 7.8×103, 2.57×103, and 1.83×103 M−1 s−1 for INH, NH, PH, and BH, respectively, imparting an important data set as a reference to the water treatment. This work may open a new door for resolving water pollution caused by INH and its derivatives.

Thermal Degradation Kinetics and pH-Rate Profile of Verbascoside and Stability Improvement by Solid Lipid Nanoparticles.

Thermal degradation of verbascoside (VB) in Acanthus ebracteatus Vahl (AE) always affects its health benefit. Here the temperature effect on VB in both AE extract and solid lipid nanoparticles (SLNs)-encapsulated AE extract was demonstrated using the Arrhenius plot. The reaction rate constants were calculated for shelf life and plotted to obtain pH-rate profiles. VB degradation was a first-order reaction. The reaction rate in a neutral to alkaline solution was faster than in an acidic solution. VB in AE extract-loaded SLNs was more stable than in uncapped AE extract. The shelf life of VB in SLNs was 153 days with activation energy (E a) of 76.16 kJ mol-1, whereas those of VB in AE extract and in AE extract solution were 75 days with E a = 78.03 kJ mol-1 and 12 days with E a = 49.24 kJ mol-1, respectively. Therefore, we anticipate that the AE extract-loaded SLNs will be beneficial for product development.

Oxidation of diclofenac by permanganate: Kinetics, products and effect of inorganic reductants

The large consumption and discharge of diclofenac (DCF) lead to its frequent detection in surface water and groundwater, posing great threats to humans and ecosystems. This study explored the oxidation kinetics of DCF by permanganate (Mn(VII)), and expounded the underlying reason for the unusual pH-dependency that was unclear in previous studies. The kinetics of DCF analogues (i.e., aromatic secondary amines) by Mn(VII) oxidation were comparatively investigated. Then, a tentative kinetic model involving the formation of an intermediate between Mn(VII) and DCF or its analogues was proposed to fit the pH-rate profile. Since DCF contained two chloro groups, and a carboxyl group which could be ionized by negative electrospray ionization, a precursor ionization scanning approach was used for the first time for detection of N-containing chlorinated oxidation products. New degradation pathways of DCF containing ring opening, carboxylation, carbonylation, electrophilic addition, hydroxylation and dehydrogenation were proposed based on the identified oxidation products. Moreover, it was demonstrated that the introduction of various reducing agents such as Mn(II), Fe(II) and bisulfite significantly improved the oxidation kinetics of DCF by Mn(VII). The positive effects of Mn(II) and Fe(II) were mainly attributed to the accelerated formation of MnO2 that acted as a catalyst or co-oxidizer contributing to DCF degradation. The presence of bisulfite caused two-stage kinetics, where a sharp drop of DCF concentration followed by a slowdown of DCF removal. In the first stage, potent reactive manganese species (e.g., Mn(III), Mn(V), and Mn(VI)) and sulfate radical were generated during reaction of bisulfite with Mn(VII), whereas bisulfite was depleted fast due to excess Mn(VII) concentrations and the system became the Mn(VII)/MnO2 system in the second stage. These results provide new insight into reaction mechanism of DCF with Mn(VII) as well as propose a feasible strategy for enhancing the treatment of DCF contaminated water by Mn(VII).

Synthesis of Oligoribonucleotides Containing a 2'-Amino-5'-S-phosphorothiolate Linkage.

Oligoribonucleotides containing a photocaged 2′-amino-5′-S-phophorothiolate linkage have potential applications as therapeutic agents and biological probes to investigate the RNA structure and function. We envisioned that oligoribonucleotides containing a 2′-amino-5′-S-phosphorothiolate linkage could provide an approach to identify the general base within catalytic RNAs by chemogenetic suppression. To enable preliminary tests of this idea, we developed synthetic approaches to a dinucleotide, trinucleotide, and oligoribonucleotide containing a photocaged 2′-amino-5′-S-phosphorothiolate linkage. We incorporated the photocaged 2′-amino-5′-S-phosphorothiolate linkage into an oligoribonucleotide substrate for the hepatitis delta virus (HDV) ribozyme and investigated the pH dependence of its cleavage following UV irradiation both in the presence and absence of the ribozyme. The substrate exhibited a pH-rate profile characteristic of the modified linkage but reacted slower when bound to the ribozyme. Cleavage inhibition by the HDV ribozyme could reflect a non-productive ground-state interaction with the modified substrate’s nucleophilic 2′-NH2 or a poor fit of the modified transition state at the ribozyme’s active site.

Structure-Activity Relationships in Nonenzymatic Template-Directed RNA Synthesis.

The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.

Beyond the Plateau: pL Dependence of Proton Inventories as a Tool for Studying Ribozyme and Ribonuclease Catalysis.

Acid/base catalysis is an important catalytic strategy used by ribonucleases and ribozymes; however, understanding the number and identity of functional groups involved in proton transfer remains challenging. The proton inventory (PI) technique analyzes the dependence of the enzyme reaction rate on the ratio of D2O to H2O and can provide information about the number of exchangeable sites that produce isotope effects and their magnitude. The Gross-Butler (GB) equation is used to evaluate H/D fractionation factors from PI data typically collected under conditions (i.e., a "plateau" in the pH-rate profile) assuming minimal change in active site residue ionization. However, restricting PI analysis to these conditions is problematic for many ribonucleases, ribozymes, and their variants due to ambiguity in the roles of active site residues, the lack of a plateau within the accessible pL range, or cooperative interactions between active site functional groups undergoing ionization. Here, we extend the integration of species distributions for alternative enzyme states in noncooperative models of acid/base catalysis into the GB equation, first used by Bevilacqua and colleagues for the HDV ribozyme, to develop a general population-weighted GB equation that allows simulation and global fitting of the three-dimensional relationship of the D2O ratio (n) versus pL versus kn/k0. Simulations using the GPW-GB equation of PI results for RNase A, HDVrz, and VSrz illustrate that data obtained at multiple selected pL values across the pL-rate profile can assist in the planning and interpreting of solvent isotope effect experiments to distinguish alternative mechanistic models.

Probing Metal-Dependent Phosphate Binding for the Catalysis of the 17E DNAzyme.

The RNA-cleaving 17E DNAzyme exhibits different levels of cleavage activity in the presence of various divalent metal ions, with Pb2+ giving the fastest cleavage. In this study, the metal-phosphate interaction is probed to understand the trend of activity with different metal ions. For the first-row transition metals, the lowest activity shown by Ni2+ correlates with the inhibition by the inorganic phosphate and its water ligand exchange rate, suggesting inner-sphere metal coordination. Cleavage activity with the two stereoisomers of the phosphorothioate-modified substrates, Rp and Sp, indicated that Mg2+, Mn2+, Fe2+, and Co2+ had the highest Sp:Rp activity ratio of >900. Comparatively, the activity was much less affected using the thiophilic metals, including Pb2+, suggesting inner-sphere coordination. The pH-rate profiles showed that Pb2+ was different than the rest of the metal ions in having a smaller slope and a similar fitted apparent pKa and the pKa of metal-bound water. Combining previous reports and our current results, we propose that Pb2+ most likely plays the role of a general acid while the other metal ions are Lewis acid catalysts interacting with the scissile phosphate.

Single Residue on the WPD-Loop Affects the pH Dependency of Catalysis in Protein Tyrosine Phosphatases.

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a noncatalytic residue in the WPD-loops of YopH and PTP1B result in shifted pH-rate profiles from an altered kinetic pKa of the nucleophilic cysteine. Compared to wild type, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations but suggest an increased preference for the WPD-loop-closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by nonchemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.