Formation Of Enzyme-substrate Complex Research Articles

Comparative Analysis of Bioelectrocatalytic Cytochrome P450 3A4 Systems

This article describes the approaches developed by the authors with the aim to increase the efficiency of electro enzymatic reactions catalyzed by cytochrome P450 3A4. A comparative analysis of cytochrome P450 3A4 systems was carried out during the formation of the functional complexes hemoprotein-flavin nucleotides as low-molecular models of NAD(P)H-dependent cytochrome P450 reductase. The formation of a productive enzyme-substrate complex before the stage ofaccepting electrons from the modified electrode was studied from the electocatalytic viewpoint. Incorporation of the enzyme into nanopores of different nature on the electrode (2D-3D transition) was also studied. The results on the electrochemical reduction of bactosomes as the functionally active models of the microsomal monooxygenase system are also considered. The electrochemical and electrocatalytic parameters of cytochrome P450 3A4 were compared for different models of the electrocatalytic generation of metabolites.

Enzymatic Performance of Aspergillus oryzae α-Amylase in the Presence of Organic Solvents: Activity, Stability, and Bioinformatic Studies.

Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme's activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents.

Biochemical and in silico evaluation of a recombinant, glucose tolerant, and highly thermostable β-glucosidase from Thermococcus radiotolerans DSM-15228

Backgroundβ-glucosidase (EC 3.2.1.21) catalyzing the β-glycosidic linkages in polysaccharides is a ubiquitous enzyme with great importance in biofuel and other industries. Enzyme inhibition by glucose has been considered as the major hurdle in the practical applications of this enzyme. Therefore, there has been a continuous search for novel β-glucosidase with high glucose tolerance and stability at industrial temperature. In the present study, recombinant of β-glucosidase from Thermococcus radiotolerans has been produced and characterized. ResultsThe enzyme was overexpressed in Escherichia coli strain BL21 (DE3) codon plus RIPL and purified by selective heat denaturation, ethanol precipitation and anion exchange chromatography. Purified enzyme displayed a band on SDS-PAGE with a molecular weight of 50 kDa. Optimum enzyme activity was found at pH 5, and 85°C, it retained more than 46% activity when incubated at 100°C for 5 min and exhibited 80% activity in the presence of 800 mM glucose. Km and Vmax values of the purified enzyme were found as 16.3 mM of pNPG and 25.8 µ moles per min. Molecular docking studies have shown a strong binding affinity of pNPG with the enzyme active site consisting of Glu365, Asn266, and Trp295 as major the active site amino acids. MD simulation analysis has shown a significantly high stability of enzyme active site and high potential of enzyme substrate complex formation. ConclusionsThe novel characteristics such as relatively low Km value, extremely high-temperature stability and tolerance of high glucose concentration advocate the enzyme as a potential candidate for the industrial applications.How to cite: Albalawi H, Altayeb HN, Iftikhar S, et al. Biochemical and in silico evaluation of a recombinant, glucose tolerant, and highly thermostable β-glucosidase from T. radiotolerans DSM-15228. Electron J Biotechnol 2023; 64. https://doi.org/10.1016/j.ejbt.2023.03.002.

Kinetic and thermodynamic characterization of keratinolytic protease from chicken feather waste degrading B. subtilis ES5

Kinetic and thermodynamic characterization of enzymes is of great important for understanding their industrial applications. The main objective of this study was to produce, purify and study the kinetic and thermodynamic properties of keratinase from B. subtilis ES5. Specific activity of the enzyme was improved by 126.16 % and 140.11 % using ammonium sulfate and dialysis purification methods, respectively. The activation energy (Ea), Gibbs free energy (∆G≠), entropy (∆S≠), enthalpy (∆H≠), free energy of substrate binding (∆G≠E−S), free energy of transition state formation (∆G≠E−T), and temperature coefficient (Q10) for casein hydrolysis were 17.59 KJ/mol, 66.11 KJ/mol, −160.88 J/mol/k, 14.95 KJ/mol, 2.61 KJ/mol, −9.21 KJ/mol, and 1.62, respectively. Thermal denaturation energy for keratinase was 110.83 KJ/mol. Kinetic studies revealed good enzyme-substrate complex formation and spontaneous conversion to product. Thermodynamic parameters indicate a relatively thermostable enzyme. These properties suggest that the present keratinase can be a potential candidate for use in different biotechnological applications.

Derivation of steady-state first-order rate constant equations for enzyme-substrate complex dissociation, as well as zero-order rate constant equations in relation to background assumptions

The maximum velocity (Vmax) of catalysis and the substrate concentration ([ST]) at half the Vmax, the KM, are regarded as steady-state (SS) parameters even though they are the outcomes of zero-order kinetics (ZOK). The research was aimed at disputing such a claim with the following objectives: To: 1) carry out an overview of issues pertaining to the validity of assumptions; 2) derive the needed steady-state (SS) equations distinct from Michaelian equations that can be fitted to both experimental variables and kinetic parameters; 3) calculate the SS first-order rate constant for the dissociation of enzyme-substrate complex (ES) to free substrate, S and enzyme, E; 4) derive the equation of rate constant as a function of the reciprocal of the duration of each catalytic event in the reaction pathway. The experimental values of the data were generated by Bernfeld and Lineweaver-Burk methods. The calculated SS 1st order-order rate constant was « the zero-order Michaelian value, and the difference is ≈ 97.59 % of the zero-order value; the SS catalytic rate differed from the zero-order catalytic rate by ≈ 76.41 % of the latter value; and it was ≈ 93.87 % with respect to the 2nd order rate constant for the formation of enzyme-substrate complex. The equations of time-dependent rate constants, KM, and dissociation constants were derived. The magnitude of [ST] must be > the concentration ([E0]) of the E for the quasi-steady-state assumption (or approximation) to hold. The SS kinetic parameters are not equivalent to zero-order parameters.

Rate constants are determinable outside the original Michaelis–Menten mathematical formalism wherein the substrate concentration range is  1.6  4.8 times enzyme concentration: A pre-steady-state scenario and beyond

For some time now, there has been growing interest in pre-steady-state (PSS) kinetic parameters for whatever reasons, the measurement of which needs high-tech equipment capable of transient time-scale duration of assay. The proposition, however, is that all kinetic parameters, PSS and beyond, can be determined with appropriate PSS derivable equations and the usual Michaelis-Menten (MM) and Briggs-Haldane (BH) equations, respectively. The objectives of the research were: 1) To derive equations, for the determination of reverse rate constant when the substrate concentration, [S] « MM constant, KM, 2) determine by calculation, the reverse rate constant, forward rate constant, and consequently, show that it is possible to determine rate constant often seen to be masked within original MM cum BH mathematical formalism, and 3) validate corollaries from the derivation that justify procedural issue. Theoretical, experimental (Bernfeld method), and computational methods were explored. Pre-steady-state equations for the determination of kinetic parameters, the reverse rate constant, k-1, for the process ES ® E + S, the 2nd order rate constant, k1, and the rate, v1, for the formation of enzyme-substrate complex, ES, were derived. The derived originating equations with associated corollaries were validated and have been seen to be capable of reproducing experimental variables and kinetic parameters; rate constants that seemed masked in MM formalism were unmasked. Steady-state (SS) cum zero order kinetic parameters were » their PSS values. “Negative” catalytic efficiency (k-1/KM) was » “positive” catalytic efficiency, (kcat/KM), with lower [ET]. In conclusion, the equations for PSS kinetic parameters were derivable. Previously masked kinetic parameters in the MM/BB mathematical formalism can now be calculated using MM data; thus, all kinetic parameters can be determined regardless of the reaction pathway's state, PSS, and SS. PSS kinetic parameters were « SS/zero order values.

Comparing similar versions of a connecting helix on the structure of Chondroitinase ABC I

Regarding the existence of similar helices on the structure of different proteins, recently, novel variants of Chondroitinase ABC I (cABC I) have been constructed, where a representative helix between two structural motifs in Chondroitinase ABC I from Proteus vulgaris has been replaced by similar versions of helices found in other proteins. The previous study has revealed that the structural features and the activity of double mutants M886A/G887E (inspired by the 30 S ribosomal protein S1 from Geminocystis herdmanii) and M889I/Q891K (inspired by the chondroitin lyase from Proteus mirabilis) is comparable with that of wild-type (WT) cABC I. Here, the kinetic parameters of the enzyme activity for the WT and double mutants were determined. Of the recombinant double mutants, M889I/Q891K gave the highest catalytic efficiency with the kcat/Km value of approximately 2.3-fold increase, as compared with the WT and M886A/G887E. Modeling of experimental data showed that the mechanism of the heat-induced structural alteration, and the enzyme-substrate complex formation, changed upon mutation. These natural versions of the connecting helix can be used as an efficient linker in protein engineering studies as well as those investigations involving the use of biological linkers.

Non-synonymous substitution of evolutionarily conserved residue in Tau class glutathione transferases alters structural and catalytic features

Plant-specific tau glutathione transferases (GSTs) are basically involved in catalysing γ-glutathione (GSH)-dependent conjugation reactions with pesticides and herbicides, which play an important role in the detoxification of pollutants. Given the lack of systematic biochemical and structural information on tau GSTs, the study of their mediated defence mechanisms against toxic compounds has been greatly hindered. Here, we reveal the importance of the Ile residue closely interacting with GSH for the structural stability and catalytic function of GST. Evolutionary conservation analysis indicated that the crucial G-site Ile55 in the SbGSTU6 was converted to Thr53 of SbGSTU7. The comparative biochemical data on SbGSTU6, SbGSTU7 and their mutants showed that the substitution of Ile by Thr caused significant decrease in the affinity and catalytic efficiency of the GSTs. The unfavourable structural flexibility and pKa distribution of the active cavity residues were also demonstrated. Crystallography studies and molecular dynamics simulations showed that the conversion resulted in the hydrogen bond recombination with GSH and conformational rearrangement of GST active cavity, in which the Ile residue was more conducive to the formation of enzyme substrate complexes. The extensive biochemical and structural data not only reveal the critical role of the conserved G-site Ile residue in catalysing GSH-conjugate reactions but also provide valuable resources for the development of GST engineering in analytical and agricultural biotechnology.

Cosolutes Modify Alkaline Phosphatase Catalysis through Osmotic Stress and Crowding Mechanisms.

Examining the effects of different cosolutes on in vitro enzyme kinetics yielded glimpses into their potential behavior when functioning in their natural, complex, in vivo milieu. Viewing cosolute in vitro influences on a model enzyme, calf intestinal alkaline phosphatase, as a combination of competitive and uncompetitive behaviors provided quantitative insights into their effects on catalysis. Observed decreases in the apparent specificity constant, Kasp, caused by the presence of polyethylene glycols or betaine in the reaction solution, indicated interference with enzyme–substrate complex formation. This competitive inhibition appeared to be driven by osmotic stress. Dextran 6 K and sucrose strongly impeded the subsequent conversion of the bound substrate into a free product, which was marked by sharp reductions in Vmax, uncompetitive inhibition. For the same step, smaller noncarbohydrate cosolutes, triethylene glycol, polyethylene glycol 400, and betaine, also behaved as uncompetitive inhibitors but to a lesser extent. However, polyethylene glycol 8000 and 20,000 were uncompetitive activators, increasing Vmax. Polyethylene glycol of molecular weight 1000 displayed intermediate effects between these two groups of noncarbohydrate cosolutes. These results suggested that crowding has a strong influence on free product formation. The combination of competitive and uncompetitive effects and mixed behaviors, caused by the cosolutes on calf intestinal alkaline phosphatase kinetics, was consistent with the trends seen in similar enzyme–cosolute studies. It is proposed that the double-displacement mechanism of alkaline phosphatases, shared by many other enzymes, could be the root of this general observation.

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors.

We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

Association-dissociation equations, distinct from Michaelis-Menten equation for the quantification of the net flux of reactants with or without immobiliser.

The formation of enzyme-substrate complex, often in connection with the adsorption of the enzyme leading to either partial immobilisation in which the enzymes are adsorbed on a colloid or total immobilisation in which the enzyme is adsorbed on a rigid immobile phase is the concern of some researchers. The interest in immobilised substrate common in biological system is not very common. The objectives of this theoretical research are the rederivation of the equations of association and dissociation of reactants in the presence of adsorbents, insoluble larger macro-or supra-molecule and elucidation of why such equations are important and generalisable. The derivations produced two different equations that describe mathematically the net flux of either the substrate where the enzyme is adsorbed or the net flux of the enzyme where the substrate is adsorbed. The derivation also produced equations of translational velocities, given the probabilities that reactions occur following complex formation or that an escape of bullet molecules or dissociation reactions occur. In conclusion two different equations need separate derivation for association and dissociation of reactants. The needs for the flux of reactants have both biological and industrial relevance, respectively due to importance of time-dependent digestive processes and for the optimisation of the production of desired products of enzymatic action. The equations describing net flux seem generalisable in that information about the physicochemical properties of both crowding agent and immobilisers may not be needed for calculations.

Synergistic effect of inhibitors (allylthiourea and 1,2,4-triazole) on the activity of wheat soil urease to reduce nitrogen loss

Urease is a nickel-dependent metalloenzyme that catalyzes the hydrolysis of urea, forming CO2 and ammonia. Urease activity in soil enables several microorganisms to utilize urea as their nitrogen source. Nitrogen loss from the soil not only reduces the yield and soil fertility, but also create adverse effects on the environment. This study investigates the combined inhibitory potential of allylthiourea +1,2,4-triazole blend on the activity of wheat soil urease and to overcome the loss of nitrogen. For this purpose, soil samples were collected from three different regions of Punjab, Pakistan, i.e., Faisalabad, Sheikhupura, and Gujranwala. The whole research work was divided into two phases. During the first phase, the effect of different inhibitors on the activity of wheat soil urease was determined. In the second phase, kinetic behavior of urease enzyme in the presence and absence of urease inhibitors was evaluated by varying several factors i.e., pH, temperature, incubation time, and substrate concentration. Results indicated a significant urease reduction after 8th-14th day of application of inhibitor’s blend. After 14 days, the effect of urease inhibitors began to decrease. Kinetic studies revealed the optimum activity of the blend around pH 6.8, temperature 35–45 ​°C and incubation time 30 ​min. The Lineweaver-Burk plot revealed that Km value increased while Vmax value significantly decreased after applying the blend because of the formation of urease inhibitor complex that further retards the formation of enzyme-substrate complex. The synergistic usage of allylthiourea and 1,2,4-triazole inhibitor’s blend can prolong the urea hydrolysis, ultimately increasing the effectiveness of fertilizers.

Enhancement in β-galactosidase activity of Streptococcus lactis cells by entrapping in microcapsules comprising of correlated silica nanoparticles

With the advent of nanotechnology there has been an impetus towards sol gel encapsulation of biocatalyst in mesoporous silica. In this study cells of Streptococcus lactis expressing β-galactosidase activity were entrapped in silica microcapsules using evaporation induced assembly through spray drying technique. These microcapsules were characterized using complementary techniques of electron microscopy and neutron/X-ray scattering. Under optimized conditions of spray drying process, the cells immobilized in silica microcapsules had higher biocatalytic activity than the free cells. Immobilized cells showed threefold increase in enzyme activity than free cells. Free cells and immobilized Streptococcus lactis cells exhibited pH optimum at 5.0 and 7.0, respectively, whereas, the temperature optimum for both the systems was observed at 45 °C. Michaelis constant for free cells and immobilized cells was 8.33 mM and 4.16 mM, respectively, while Vmax for free cells and immobilized cells was 71.43 μmoles min−1 and 125 μmoles min−1, respectively. The decrease in apparent Km is correlated with the fold increase in apparent Vmax and can be interpreted as favorable for enzyme substrate complex formation when cells were entrapped in microcapsules. Herein, the concept of immobilization using spray dryer at optimal inlet temperature has emerged as an efficient mode for enhancing the catalytic activity of the microencapsulated microbial cells and a plausible mechanism is suggested.

Nitrogen acquisition from mineral-associated proteins by an ectomycorrhizal fungus.

In nitrogen (N)-limited boreal forests, trees depend on the decomposing activity of their ectomycorrhizal (ECM) fungal symbionts to access soil N. A large fraction of this N exists as proteinaceous compounds associated with mineral particles. However, it is not known if ECM fungi can access these mineral-associated proteins; accordingly, possible acquisition mechanisms have not been investigated. With tightly controlled isotopic, spectroscopic, and chromatographic experiments, we quantified and analyzed the mechanisms of N acquisition from iron oxide mineral-associated proteins by Paxillus involutus, a widespread ECM fungus in boreal forests. The fungus acquired N from the mineral-associated proteins. The collective results indicated a proteolytic mechanism involving formation of the crucial enzyme-substrate complexes at the mineral surfaces. Hence, the enzymes hydrolyzed the mineral-associated proteins without initial desorption of the proteins. The proteolytic activity was suppressed by adsorption of proteases to the mineral particles. This process was counteracted by fungal secretion of mineral-surface-reactive compounds that decreased the protease-mineral interactions and thereby promoted the formation of enzyme-substrate complexes. The ability of ECM fungi to simultaneously generate extracellular proteases and surface-reactive metabolites suggests that they can play an important role in unlocking the large N pool of mineral-associated proteins to trees in boreal forests.

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases.

Nucleoside phosphorylases catalyze the reversible phosphorolysis of nucleosides to heterocyclic bases, giving α-d-ribose-1-phosphate or α-d-2-deoxyribose-1-phosphate. These enzymes are involved in salvage pathways of nucleoside biosynthesis. The level of these enzymes is often elevated in tumors, which can be used as a marker for cancer diagnosis. This review presents the analysis of conformations of nucleosides and their analogues in complexes with nucleoside phosphorylases of the first (NP-1) family, which includes hexameric and trimeric purine nucleoside phosphorylases (EC 2.4.2.1), hexameric and trimeric 5′-deoxy-5′-methylthioadenosine phosphorylases (EC 2.4.2.28), and uridine phosphorylases (EC 2.4.2.3). Nucleosides adopt similar conformations in complexes, with these conformations being significantly different from those of free nucleosides. In complexes, pentofuranose rings of all nucleosides are at the W region of the pseudorotation cycle that corresponds to the energy barrier to the N↔S interconversion. In most of the complexes, the orientation of the bases with respect to the ribose is in the high-syn region in the immediate vicinity of the barrier to syn ↔ anti transitions. Such conformations of nucleosides in complexes are unfavorable when compared to free nucleosides and they are stabilized by interactions with the enzyme. The sulfate (or phosphate) ion in the active site of the complexes influences the conformation of the furanose ring. The binding of nucleosides in strained conformations is a characteristic feature of the enzyme–substrate complex formation for this enzyme group.

Tyr320 is a molecular determinant of the catalytic activity of β-glucosidase from Neosartorya fischeri

β-Glucosidases (BGL) are key members of the cellulase enzyme complex that determine efficiency of lignocellulosic biomass degradation, which have shown great functional importance to many biotechnological systems. A previous reported BGL from Neosartorya fischeri (NfBGL) showed much higher activity than other BGLs. Screening the important residues based on sequence alignment, analyzing a homology model, and subsequent alteration of individually screened residues by site-directed mutagenesis were carried out to investigate the molecular determinants of the enzyme's high catalytic efficiency. Tyr320, located in the wild-type NfBGL substrate-binding pocket was identified as crucial to the catalytic function of NfBGL. The replacement of Tyr320 with aromatic amino acids did not significantly alter the catalytic efficiency towards p-nitrophenyl β-d-glucopyranoside (pNPG). However, mutants with charged and hydrophilic amino acids showed almost no activity towards pNPG. Computational studies suggested that an aromatic acid is required at position 320 in NfBGL to stabilize the enzyme-substrate complex formation. This knowledge on the mechanism of action of the molecular determinants can also help rational protein engineering of BGLs.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes.

Conformational Dynamics of Dioxygenase AlkB and DNA in the Course of Catalytically Active Enzyme–Substrate Complex Formation

Fe2+/2-ketoglutarate-dependent DNA-dioxygenase AlkB from Escherichia coli is able to restore the native structure of alkylated DNA bases. The enzymatic process utilizes the molecular oxygen, and proceeds through a mechanism of oxidative dealkylation. Here, the kinetics of conformational changes of AlkB and DNA substrates in the course of binding steps were studied. Nickel and cobalt divalent ions were used instead of Fe2+ as metal cofactors in order to inhibit the catalytic activity of AlkB and to study certain stages leading to the formation of a catalytically active enzyme–substrate complex.

Conserved residue His-257 of Vibrio cholerae flavin transferase ApbE plays a critical role in substrate binding and catalysis

The flavin transferase ApbE plays essential roles in bacterial physiology, covalently incorporating FMN cofactors into numerous respiratory enzymes that use the integrated cofactors as electron carriers. In this work we performed a detailed kinetic and structural characterization of Vibrio cholerae WT ApbE and mutants of the conserved residue His-257, to understand its role in substrate binding and in the catalytic mechanism of this family. Bi-substrate kinetic experiments revealed that ApbE follows a random Bi Bi sequential kinetic mechanism, in which a ternary complex is formed, indicating that both substrates must be bound to the enzyme for the reaction to proceed. Steady-state kinetic analyses show that the turnover rates of His-257 mutants are significantly smaller than those of WT ApbE, and have increased Km values for both substrates, indicating that the His-257 residue plays important roles in catalysis and in enzyme-substrate complex formation. Analyses of the pH dependence of ApbE activity indicate that the pKa of the catalytic residue (pKES1) increases by 2 pH units in the His-257 mutants, suggesting that this residue plays a role in substrate deprotonation. The crystal structures of WT ApbE and an H257G mutant were determined at 1.61 and 1.92 Å resolutions, revealing that His-257 is located in the catalytic site and that the substitution does not produce major conformational changes. We propose a reaction mechanism in which His-257 acts as a general base that deprotonates the acceptor residue, which subsequently performs a nucleophilic attack on FAD for flavin transfer.

Modulation of Kinetic Pathways of Enzyme-Substrate Interaction in a Microfluidic Channel: Nanoscopic Water Dynamics as a Switch.

Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles.