Double-displacement Mechanism Research Articles

Cooperative Diarylborinic Acid/Chloride-Catalyzed Formal SNi Reaction of cis-4-Hydroxymethyl-1,2-Cyclopentene Oxides.

A catalytic formal SNi reaction was designed to achieve stereoretentive products for cis-4-hydroxymethyl-1,2-cyclopentene oxides by using diarylborinic acid as a dual role catalyst and chloride as a catalytic transient nucleophile through a double-displacement mechanism. This reaction offers the advantages of a low catalyst loading of 0.1 mol % and wide substrate scope, even including N-substituents. The use of chiral boron acid as a catalyst for this reaction was also attempted.

Bimodal substrate binding in the active site of the glycosidase BcX.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.

Molecular Mechanism of Double-Displacement Retaining β-Kdo Glycosyltransferase WbbB.

Glycosyltransferases (GTs) are pivotal enzymes involved in glycosidic bond synthesis, which can lead to either retention or inversion of the glycosyl moiety's anomeric configuration. However, the catalytic mechanism for retaining GTs remains a subject of controversy. In this study, we employ MD and QM/MM metadynamics to investigate the double-displacement catalytic mechanism of the retaining β-Kdo transferase WbbB. Our findings demonstrate that the nucleophile Asp232 initiates the reaction by attacking the sugar ring containing a carboxylate at the anomeric position, forming a covalent adduct. Subsequently, the adduct undergoes a rotational rearrangement, ensuring proper orientation of the anomeric carbon for the acceptor substrate. In the second step, Glu158 acts as the catalytic base to abstract the proton of the acceptor substrate to complete the transglycosylation reaction. Notably, His265 does not function as the anticipated catalytic acid; instead, it stabilizes the phosphate group through H-bonding interactions. Our simulations support the double-displacement mechanism implicated from the crystallographic studies of WbbB. This mechanism deviates from the common SNi-type and retaining glycoside hydrolase mechanisms, thereby expanding our understanding of GT catalytic mechanisms.

An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans

BackgroundCandida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking.ResultsWe describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein’s identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and β-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports.ConclusionsThe methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug’s development.

Substitution of acidic residues near the catalytic Glu131 leads to human HYAL1 activity at neutral pH via charge-charge interactions.

Human hyaluronidase 1 (HYAL1) and PH20 play vital roles in degrading hyaluronic acids through the substrate-assisted double displacement mechanism. While HYAL1, a lysosomal enzyme, functions optimally under acidic conditions, PH20, a sperm surface hyaluronidase, displays a broader pH range, from acidic to neutral. Our objective was to extend HYAL1's pH range towards neutral pH by introducing repulsive charge-charge interactions involving the catalytic Glu131, increasing its pKa as the proton donor. Substituting individual acidic residues in the β3-loop (S77D), β3'-β3″ hairpin (T86D and P87E), and at Ala132 (A132D and A132E) enabled HYAL1 to demonstrate enzyme activity at pH 7, with the mutants S77D, P87E, and A132E showing the highest activity in the substrate gel assay. However, double and triple substitutions, including S77D/T86D/A132E as found in the PH20 configuration, did not result in enhanced activity compared to single substitutions. Conversely, PH20 mutants with non-acidic substitutions, such as D94S in the β3-loop and D103T in the β3'-β3″ hairpin, significantly reduced activity within the pH range of 4 to 7. However, the PH20 mutant E149A, reciprocally substituted compared to A132E in HYAL1, exhibited activity similar to PH20 wild-type (WT) at pH 7. In a turbidimetric assay, HYAL1 mutants with single acidic substitutions exhibited activity similar to that of PH20 WT at pH 7. These results suggest that substituting acidic residues near Glu131 results in HYAL1 activity at neutral pH through electrostatic repulsion. This study highlights the significance of charge-charge interactions in both HYAL1 and PH20 in regulating the pH-dependent activity of hyaluronidases.

Preorganized Internal Electric Field Promotes a Double-Displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase.

Adenine DNA glycosylase (MutY) is a monofunctional glycosylase, removing adenines (A) misinserted opposite 8-oxo-7,8-dihydroguanine (OG), a common product of oxidative damage to DNA. Through multiscale calculations, we decipher a detailed adenine excision mechanism of MutY that is consistent with all available experimental data, involving an initial protonation step and two nucleophilic displacement steps. During the first displacement step, N-glycosidic bond cleavage is accompanied by the attack of the carboxylate group of residue Asp144 at the anomeric carbon (C1'), forming a covalent glycosyl-enzyme intermediate to stabilize the fleeting oxocarbenium ion. After departure of the excised base, water nucleophiles can be recruited to displace Asp144, completing the catalytic cycle with retention of stereochemistry at the C1' position. The two displacement reactions are found to mostly involve the movement of the oxocarbenium ion, occurring with large charge reorganization and thus sensitive to the internal electric field (IEF) exerted by the polar protein environment. Intriguingly, we find that the negatively charged carboxylate group is a good nucleophile for the oxocarbenium ion, yet an unactivated water molecule is not, and that the electric field catalysis strategy is used by the enzyme to enable its unique double-displacement reaction mechanism. A strong IEF, pointing toward 5' direction of the substrate sugar ring, greatly facilitates the second displacement reaction at the expense of elevating the barrier of the first one, thereby allowing both reactions to occur. These findings not only increase our understanding of the strategies used by DNA glycosylases to repair DNA lesions, but also have important implications for how internal/external electric field can be applied to modulate chemical reactions.

Trapping and retaining intermediates in glycosyltransferases

Glycosyltransferases (GTs) attach sugar molecules to a broad range of acceptors, generating a remarkable amount of structural diversity in biological systems. GTs are classified as either 'retaining' or 'inverting' enzymes. Most retaining GTs typically use an SNi mechanism. In a recent article in the JBC, Doyle et al. demonstrates a covalent intermediate in the dual-module KpsC GT (GT107) supporting a double displacement mechanism.

Mechanism and linkage specificities of the dual retaining β-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-β-d-manno-oct-2-ulosonic acid (β-Kdo) from cytidine-5'-monophospho-β-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with β-(2→4) and β-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining β-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the β-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/β domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

A Review on the Various Sources of β-Galactosidase and Its Lactose Hydrolysis Property.

β-Galactosidase is a glycoside hydrolase enzyme that possesses both hydrolytic and transgalactosylation properties and has several benefits and advantages in the food and dairy industries. The catalytic process of β-galactosidase involves the transfer of a sugar residue from a glycosyl donor to an acceptor via a double-displacement mechanism. Hydrolysis prevails when water acts as an acceptor, resulting in the production of lactose-free products. Transgalactosylation prevails when lactose acts as an acceptor, resulting in the production of prebiotic oligosaccharides. β-Galactosidase is also obtained from many sources including bacteria, yeast, fungi, plants, and animals. However, depending on the origin of the β-galactosidase, the monomer composition and their bonds may differ, thereby influencing their properties and prebiotic efficacy. Thus, the increasing demand for prebiotics in the food industry and the search for new oligosaccharides have compelled researchers to search for novel sources of β-galactosidase with diverse properties. In this review, we discuss the properties, catalytic mechanisms, various sources and lactose hydrolysis properties of β-galactosidase.

In‐Situ Capillary Pressure and Its Interrelationships With Flow Characteristics During Steady‐State Three‐Phase Flow in Water‐Wet Berea Sandstone

AbstractWe present a detailed multi‐level investigation of steady‐state three‐phase flow processes in a water‐wet Berea sandstone sample. We use high‐resolution micro‐computed tomography images acquired during micro‐scale core‐flooding experiments which include successive gas injection, waterflooding, and nonspreading oil flooding steps. We develop and employ robust techniques to comprehensively characterize and probe the complex interrelationships between the pore‐scale three‐phase capillary pressures, fluid occupancy, displacement events, and the macroscopic flow behavior. The results demonstrate that the local capillary pressures involving the invading phase rise or drop in compliance with the changes in the macroscopic flow ratios. For instance, the gas‐oil capillary pressures were at their highest levels during the gas injection process (highest gas/oil fractional flow) and dropped during the subsequent processes. More importantly, the oil‐water capillary pressure is found to sharply rise during the gas injection process, implying that the gas‐displacing‐oil‐displacing‐water double displacements are the dominant double displacement mechanism during this experiment. For the waterflooding test, the local gas‐oil capillary pressures consistently decrease due to water‐displacing‐oil‐displacing‐gas double displacement events. Moreover, the results show that the variations in the gas‐liquid capillary pressures are more significant than those in the oil‐water capillary pressures. Also, the variations in all capillary pressures during gas injection are higher than in the subsequent processes. The higher variations in capillary pressure values are attributed to more localized and less uniform pore‐scale events across the medium. Finally, the capillary pressure gradients along the flow direction are incorporated in the three‐phase relative permeability measurements.

The retaining β-Kdo glycosyltransferase WbbB uses a double-displacement mechanism with an intermediate adduct rearrangement step

WbbB, a lipopolysaccharide O-antigen synthesis enzyme from Raoultella terrigena, contains an N-terminal glycosyltransferase domain with a highly modified architecture that adds a terminal β-Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue to the O-antigen saccharide, with retention of stereochemistry. We show, using mass spectrometry, that WbbB forms a covalent adduct between the catalytic nucleophile, Asp232, and Kdo. We also determine X-ray structures for the CMP-β-Kdo donor complex, for Kdo-adducts with D232N and D232C WbbB variants, for a synthetic disaccharide acceptor complex, and for a ternary complex with both a Kdo-adduct and the acceptor. Together, these structures show that the enzyme-linked Asp232-Kdo adduct rotates to reposition the Kdo into a second sub-site, which then transfers Kdo to the acceptor. Retaining glycosyltransferases were thought to use only the front-side SNi substitution mechanism; here we show that retaining glycosyltransferases can also potentially use double-displacement mechanisms, but incorporating an additional catalytic subsite requires rearrangement of the protein’s architecture.

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.

Exploring the catalytic mechanism of a novel β-glucosidase BGL0224 from Oenococcus oeni SD-2a: Kinetics, spectroscopic and molecular simulation

The β-glucosidase derived from microorganisms has attracted worldwide interest for their industrial applications, but studies on β-glucosidases from Oenococcus oeni are rare. In this paper, catalytic mechanism of a novel β-glucosidase BGL0224 of Oenococcus oeni SD-2a was explored for the first time by kinetic parameters determination, fluorescence spectroscopy and quenching mechanism analysis, molecular dynamics simulation. The results indicated that BGL0224 had universal catalytic effect on different types of glycoside substrates, but the catalytic efficiencies were different. Fluorescence quenching analysis results suggested that the quenching processes between BGL0224 and seven kinds of substrates were predominated by the static quenching mechanism. A reasonable three-dimensional model of BGL0224 was obtained using the crystal structure of E.coli BglA as a template. The analysis results of molecular simulation (RMSD, Rg, RMSF and hydrogen bonding) showed that the composite system ‘BGL0224-pNPG’ was very stable after 40 ns. The catalytic process of BGL0224 acting on ‘p-Nitrophenyl β-d-glucopyranoside’ conformed to the double displacement mechanism. Two glutamic acid residues ‘Glu178 and Glu377’ played a vital role in the whole catalytic process. Overall, this study gave specific insights on the catalytic mechanism of BGL0224, which was of great significance for developing its potential applications in food industry.

Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-β-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the -1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.

The Structurally Unusual Retaining β‐Kdo Glycosyltransferase WbbB Uses a Double‐Displacement Mechanism with an Intermediate Adduct Rearrangement Step

WbbB is a modular, trifunctional glycosyltransferase (GT) which synthesizes lipopolysaccharide O‐antigen in Raoultella terrigena. The two C‐terminal WbbB GT domains build the polysaccharide repeat, while the N‐terminal GT domain acts as a terminator, adding a β‐Kdo (3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid) residue to O3 to rhamnose as required for O‐antigen export. This N‐terminal GT domain is highly unusual in sequence and was not recognized as a GT by bioinformatics tools. The structure shows the dual Rossmann‐fold motifs characteristic of GT‐Bs, but with extensive deletions, insertions and rearrangements result in a unique architecture. The CMP‐binding site, however, retains motifs homologous to GT‐B sialyltransferases. WbbB is a retaining GT, transferring Kdo from the CMP‐®‐Kdo donor with net retention of anomeric configuration. All well‐characterized retaining GTs seem to employ a front‐side SNi substitution mechanism. This is somewhat surprising, as analogous retaining glycosyl hydrolases use a double‐displacement mechanism; here an essential catalytic acid residue attacks the donor saccharide to form a covalently bonded intermediate, with a second acidic residue acting as a general base to hydrolyse this adduct. We show, using mass spectrometry, that WbbB forms a Kdo adduct with Asp232 and its variants (D232N or D232C), while a D232A variant is both wholly inactive and forms no adduct. The x‐ray structure of D232N CMP‐®‐Kdo donor complex shows that the anomeric carbon of the donor is inaccessible to any potential acceptor but is instead positioned immediately in contact with, and in line with, the carboxylate of Asp232. Structures of D232N‐ and D232C‐Kdo adducts show that the Kdo adduct is rearranged into a second half‐site, interacting with a distinct set of catalytic residues. A ternary complex of D232C‐Kdo plus a synthetic acceptor shows that Glu158 forms direct hydrogen bonds with O3 of rhamnose, positioned adjacent to and in‐line with the anomeric carbon of Kdo, activating it for attack. Glu158 is also essential, with variants being wholly inactive. Together, this shows that WbbB uses a glycosyl hydrolase‐like double‐displacement mechanism. We propose that crowding around the anomeric carbon by the carboxylate group likely precludes retaining ulosonic acid transferases using the more straightforward SNi mechanism, while adduct rearrangement avoids having the acceptor compete with the leaving nucleotide for access to the anomeric carbon of the donor.Support or Funding InformationThis work was funded by an NSERC Discovery grant (04045‐2015) to MSK and support from the Canadian Glycomics Network (SD‐1) to TLL; TJBF is the recipient of a University of Guelph Graduate Excellence Entrance Scholarship and an Ontario Graduate Scholarship.

In situ pore-scale analysis of oil recovery during three-phase near-miscible CO2 injection in a water-wet carbonate rock

We study in situ three-phase near-miscible CO2 injection in a water-wet carbonate rock at elevated temperature and pressure using X-ray microtomography. We examine the recovery mechanisms, presence or absence of oil layers, pore occupancy and interfacial areas during a secondary gas injection process. In contrast to an equivalent immiscible system, we did not observe layers of oil sandwiched between gas in the centre of the pore space and water in the corners. At near-miscible conditions, the measured contact angle between oil and gas was approximately 73°, indicating only weak oil wettability in the presence of gas. Oil flows in the centres of large pores, rather than in layers for immiscible injection, when displaced by gas. This allows for a rapid production of oil since it is no longer confined to movement in thin layers. A significant recovery factor of 80% was obtained and the residual oil saturation existed as disconnected blobs in the corners of the pore space. At equilibrium, gas occupied the biggest pores, while oil and water occupied pores of varying sizes (small, medium and large). Again, this was different from an immiscible system, where water occupied only the smallest pores. We suggest that a double displacement mechanism, where gas displaces water that displaces oil is responsible for shuffling water into larger pores than that seen after initial oil injection. This is only possible since, in the absence of oil layers, gas can contact water directly. The gas-oil and oil-water interfacial areas are lower than in the immiscible case, since there are no oil layers and even water layers in the macro-pore space become disconnected; in contrast, there is a larger direct contact of oil to the solid. These results could serve as benchmarks for developing near-miscible pore-scale modelling tools.

Geometric considerations support the double-displacement catalytic mechanism of l-asparaginase.

Twenty crystal structures of the complexes of l-asparaginase with l-Asn, l-Asp, and succinic acid that are currently available in the Protein Data Bank, as well as 11 additional structures determined in the course of this project, were analyzed in order to establish the level of conservation of the geometric parameters describing interactions between the substrates and the active site of the enzymes. We found that such stereochemical relationships are highly conserved, regardless of the organism from which the enzyme was isolated, specific crystallization conditions, or the nature of the ligands. Analysis of the geometry of the interactions, including Bürgi-Dunitz and Flippin-Lodge angles, indicated that Thr12 (Escherichia coli asparaginase II numbering) is optimally placed to be the primary nucleophile in the most likely scenario utilizing a double-displacement mechanism, whereas catalysis through a single-displacement mechanism appears to be the least likely.

Computational and Enzymatic Analyses Unveil the Catalytic Mechanism of Thermostable Trehalose Synthase and Suggest Strategies for Improved Bioconversion.

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, and is therefore essential for trehalose production. Consequently, dissecting the catalytic mechanism of TreS is important for enzyme optimization and industrial applications. TreS from Thermobaculum terrenum (TtTreS) is a thermostable enzyme. Here, we studied the composition of the TtTreS active site through computer calculation and enzyme analysis. The results were consistent with a two-step double-displacement mechanism, similar to that of glycoside hydrolase 13 family enzymes. However, our data suggested that glucose rotation, following breakage of the α-1,4 glycosidic bond, is a key factor determining the reaction direction and conversion rate. The N246 residue plays an important role in glucose rotation. Moreover, we established a saturated mutation model for the nonconserved amino acids around the substrate gateway domain. Finally, four TtTreS mutants (K136T, Y137D, K138N, and D139S) resulted in improved trehalose yield compared to that of the wild-type enzyme.

The pKa values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme.

T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H-T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl-enzyme intermediate (Kuroki et al., PNAS 1999; 96:8949-8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the putative nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H-T4L follows a reverse protonation pathway in which only a minor population of the free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H-T4L, including the active site Arg145, are positively charged under neutral pH conditions. BRIEF STATEMENT: The replacement of a single amino acid changes T4 lysozyme from an inverting to a retaining glycoside hydrolase. Using NMR spectroscopy, we measured the pKa values of the ionizable residues in the active site of this mutant enzyme. Along with previously reported data, these results provide important constraints for understanding the catalytic mechanisms by which the wild-type and mutant form of T4 lysozyme cleave bacterial peptidoglycan.

Modulating the Nucleophile of a Glycoside Hydrolase through Site-Specific Incorporation of Fluoroglutamic Acids.

Understanding the detailed mechanisms of enzyme-catalyzed hydrolysis of the glycosidic bond is fundamentally important, not only to the design of tailored cost-efficient, stable and specific catalysts but also to the development of specific glycosidase inhibitors as therapeutics. Retaining glycosidases employ two key carboxylic acid residues, typically glutamic acids, in a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. One Glu functions as a nucleophile while the other acts as a general acid/base. A significant part of enzymatic proficiency is attributed to a "perfect match" of the electrostatics provided by these key residues, a hypothesis that has been remarkably difficult to prove in model systems or in enzymes themselves. We experimentally probe this synergy by preparing synthetic variants of a model glycosidase Bacillus circulans β-xylanase (Bcx) with the nucleophile Glu78 substituted by 4-fluoro or 4,4-difluoroglutamic acid to progressively reduce nucleophilicity. These Bcx variants were semisynthesized by preparation of optically pure fluoroglutamic acid building blocks, incorporation into synthetic peptides, and ligation onto a truncated circular permutant of Bcx. By measuring the effect of altered electrostatics in the active site on enzyme kinetic constants, we show that lowering the nucleophile p Ka by two units shits the pH-dependent activity by one pH unit. Linear free energy correlations using substrates of varying leaving group ability indicate that by reducing nucleophilic catalysis the concerted mechanism of the enzyme is disrupted and shifted toward a dissociative pathway. Our study represents the first example of site-specific introduction of fluorinated glutamic acids into any protein. Furthermore, it provides unique insights into the synergy of nucleophilic and acid/base catalysis within an enzyme active site.