Investigating the Effect of Two-Point Surface Attachment on Enzyme Stability and Activity.

Immobilization on solid supports provides an effective way to improve enzyme stability and simplify downstream processing for biotechnological applications, which has been widely used in research and in applications. However, surface immobilization may disrupt enzyme structure due to interactions between the enzyme and the supporting substrate, leading to a loss of the enzyme catalytic efficiency and stability. Here, we use a model enzyme, nitroreductase (NfsB), to demonstrate that engineered variants with two strategically positioned surface-tethering sites exhibit improved enzyme stability when covalently immobilized onto a surface. Tethering sites were designed based on molecular dynamics (MD) simulations, and enzyme variants containing cysteinyl residues at these positions were expressed, purified, and immobilized on maleimide-terminated self-assembled monolayer (SAM) surfaces. Sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to deduce the NfsB enzyme orientations, which were found to be consistent with those predicted from the MD simulations. Thermal stability analyses demonstrated that NfsB variants immobilized through two tethering sites exhibited generally improved thermal stability compared with enzymes tethered at only one position. For example, NfsB enzyme chemically immobilized via positions 423 and 111 exhibits at least 60% stability increase compared to chemically immobilized NfsB mutant via a single site. This research develops a generally applicable and systematic approach using a combination of simulation and experimental methods to rationally select protein immobilization sites for the optimization of surface-immobilized enzyme activity and stability.

In planta variation of volatile biosynthesis: an alternative biosynthetic route to the formation of the pathogen-induced volatile homoterpene DMNT via triterpene degradation in Arabidopsis roots.

Plant-derived volatile compounds such as terpenes exhibit substantial structural variation and serve multiple ecological functions. Despite their structural diversity, volatile terpenes are generally produced from a small number of core 5- to 20-carbon intermediates. Here, we present unexpected plasticity in volatile terpene biosynthesis by showing that irregular homo/norterpenes can arise from different biosynthetic routes in a tissue specific manner. While Arabidopsis thaliana and other angiosperms are known to produce the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) or its C16-analog (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene by the breakdown of sesquiterpene and diterpene tertiary alcohols in aboveground tissues, we demonstrate that Arabidopsis roots biosynthesize DMNT by the degradation of the C30 triterpene diol, arabidiol. The reaction is catalyzed by the Brassicaceae-specific cytochrome P450 monooxygenase CYP705A1 and is transiently induced in a jasmonate-dependent manner by infection with the root-rot pathogen Pythium irregulare. CYP705A1 clusters with the arabidiol synthase gene ABDS, and both genes are coexpressed constitutively in the root stele and meristematic tissue. We further provide in vitro and in vivo evidence for the role of the DMNT biosynthetic pathway in resistance against P. irregulare. Our results show biosynthetic plasticity in DMNT biosynthesis in land plants via the assembly of triterpene gene clusters and present biochemical and genetic evidence for volatile compound formation via triterpene degradation in plants.

Simultaneous Observation of the Orientation and Activity of Surface-Immobilized Enzymes.

Surface immobilized enzymes have been widely used in many applications such as biosensors, biochips, biofuel production, and biofuel cell construction. Many factors dictate how enzymes' structure, activity, and stability may change when immobilized, including surface functionalization, immobilization chemistry, nature of the solid support, and enzyme surface density. To better understand how immobilization affects enzyme structure and activity, we have developed a method to measure both surface-sensitive protein vibrational spectra and enzymatic activity simultaneously. To accomplish this, an optical/fluorescence microscope was incorporated into a sum frequency generation (SFG) spectrometer. Using ß-glucosidase (ß-Glu) as a model system, enzymes were covalently tethered to a self-assembled monolayer surface using cysteine-maleimide chemistry. Their orientations were determined by SFG spectroscopy, with a single native cysteine residue oriented toward the functionalized surface, and activity measured simultaneously using a fluorogenic substrate resorufin ß-d-glucopyranoside, with a loss of activity of 53% as compared to comparable solution measurements. Measuring ß-Glu activity and orientation simultaneously provides more accurate information for designing and further improving enzymatic activity of surface-bound enzymes.

Engineered Surface-Immobilized Enzyme that Retains High Levels of Catalytic Activity in Air.

In the absence of aqueous buffer, most enzymes retain little or no activity; however, "water-free" enzymes would have many diverse applications. Here, we describe the chemically precise immobilization of an enzyme on an engineered surface designed to support catalytic activity in air at ambient humidity. Covalent immobilization of haloalkane dehalogenase on a surface support displaying poly(sorbitol methacrylate) chains resulted in ∼40-fold increase in activity over lyophilized enzyme powders for the gas-phase dehalogenation of 1-bromopropane. The activity of the immobilized enzyme in air approaches 25% of the activity obtained in buffer for the immobilized enzyme. Poly(sorbitol methacrylate) appears to enhance activity by replacing protein-water interactions, thereby preserving the protein structure.

Symmetry-Directed Self-Assembly of a Tetrahedral Protein Cage Mediated by de Novo-Designed Coiled Coils.

The organization of proteins into new hierarchical forms is an important challenge in synthetic biology. However, engineering new interactions between protein subunits is technically challenging and typically requires extensive redesign of protein-protein interfaces. We have developed a conceptually simple approach, based on symmetry principles, that uses short coiled-coil domains to assemble proteins into higher-order structures. Here, we demonstrate the assembly of a trimeric enzyme into a well-defined tetrahedral cage. This was achieved by genetically fusing a trimeric coiled-coil domain to its C terminus through a flexible polyglycine linker sequence. The linker length and coiled-coil strength were the only parameters that needed to be optimized to obtain a high yield of correctly assembled protein cages.

Effect of Surface Crowding and Surface Hydrophilicity on the Activity, Stability and Molecular Orientation of a Covalently Tethered Enzyme.

We have investigated two surface properties that are generally thought to have an important influence of enzyme activity and stability: surface hydrophobicity and surface crowding. Here two variants of an engineered bacterial nitro-reductase were covalently tethered to orient the protein's pseudo-2-fold symmetry axis either parallel or perpendicular to the surface. The surface hydrophobicity was systematically varied by changing the ratio of methyl- to hydroxyl-groups displayed on the SAM surface, and the effects on enzyme activity, thermal stability, and structure investigated. Increasing surface hydrophobicity progressively decreased enzyme activity, but had no effect on thermal stability. Surface-sensitive sum frequency generation and attenuated total reflectance Fourier transform IR spectroscopies indicated that the enzyme is not denatured by the more hydrophobic surface, but is more likely trapped in less active conformations by transient hydrophobic interactions. In contrast, increasing enzyme surface concentration increased the specific activity of the parallel oriented enzyme, but had no effect on the activity of the perpendicularly oriented enzyme, suggesting that crowding effects are highly context dependent.

Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

Mechanism of N-hydroxylation catalyzed by flavin-dependent monooxygenases.

Aspergillus fumigatus siderophore (SidA), a member of class B flavin-dependent monooxygenases, was selected as a model system to investigate the hydroxylation mechanism of heteroatom-containing molecules by this group of enzymes. SidA selectively hydroxylates ornithine to produce N(5)-hydroxyornithine. However, SidA is also able to hydroxylate lysine with lower efficiency. In this study, the hydroxylation mechanism and substrate selectivity of SidA were systematically studied using DFT calculations. The data show that the hydroxylation reaction is initiated by homolytic cleavage of the O-O bond in the C(4a)-hydroperoxyflavin intermediate, resulting in the formation of an internal hydrogen-bonded hydroxyl radical (HO(•)). As the HO(•) moves to the ornithine N(5) atom, it rotates and donates a hydrogen atom to form the C(4a)-hydroxyflavin. Oxygen atom transfer yields an aminoxide, which is subsequently converted to hydroxylamine via water-mediated proton shuttling, with the water molecule originating from dehydration of the C(4a)-hydroxyflavin. The selectivity of SidA for ornithine is predicted to be the result of the lower energy barrier for oxidation of ornithine relative to that of lysine (16 vs 24 kcal/mol, respectively), which is due to the weaker stabilizing hydrogen bond between the incipient HO(•) and O3' of the ribose ring of NADP(+) in the transition state for lysine.

Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

C4a-hydroperoxyflavin formation in N-hydroxylating flavin monooxygenases is mediated by the 2'-OH of the nicotinamide ribose of NADP⁺.

Flavin-dependent monooxygenases must stabilize a C4a-hydroperoxyflavin intermediate to hydroxylate their respective substrates. Formation and decay of the C4a-hydroperoxyflavin were monitored under rapid reaction kinetic conditions in SidA, an N-hydroxylating monooxygenase involved in siderophore biosynthesis. Solvent kinetic isotope effect studies of flavin oxidation indicate that both hydrogen peroxide elimination and water elimination occur via abstraction of hydrogen from the N5 of the flavin. Kinetic isotope effect and density functional theory results are consistent with the transfer of a proton from the 2'-OH of the nicotinamide ribose of nicotinamide adenine dinucleotide phosphate (NADP⁺) to the C4a-peroxyflavin to form the C4a-hydroperoxyflavin. This represents a novel role for NADP⁺ in the reaction of flavin-dependent enzymes.

Tryptophan-47 in the active site of Methylophaga sp. strain SK1 flavin-monooxygenase is important for hydride transfer.

Flavin-dependent monooxygenase (FMO) from Methylophaga sp. strain SK1 catalyzes the NADPH- and oxygen-dependent hydroxylation of a number of xenobiotics. Reduction of the flavin cofactor by NADPH is required for activation of molecular oxygen. The role of a conserved tryptophan at position 47 was probed by site-directed mutagenesis. FMOW47A resulted in an insoluble inactive protein; in contrast, FMOW47F was soluble and active. The spectrum of the flavin in the mutant enzyme was redshifted, indicating a change in the flavin environment. The kcat values for NADPH, trimethylamine, and methimazole, decreased 5-8-fold. Primary kinetic isotope effect values were higher, indicating that hydride transfer is more rate-limiting in the mutant enzyme. This is supported by a decrease in the rate constant for flavin reduction and in the solvent kinetic isotope effect values. Results from molecular dynamics simulations show reduced flexibility in active site residues and, in particular, the nicotinamide moiety of NADP+ in FMOW47F. This was supported by thermal denaturation experiments. Together, the data suggests that W47 plays a role in maintaining the overall protein flexibility that is required for conformational changes important in hydride transfer.

Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis.

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant-plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C(20)-precursor (E,E)-geranyllinalool to the insect-induced C(16)-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C(15)-analog (E)-nerolidol, which is converted to the respective C(11)-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

HIV-1 binds dynein directly to hijack microtubule transport machinery.

Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet the molecular mechanism that describes how viruses connect to the motor machinery is unknown. Here, we demonstrate the first example of viral microtubule trafficking from purified components: HIV-1 hijacking microtubule transport machinery. We discover that HIV-1 directly binds to the retrograde microtubule-associated motor, dynein, and not via a cargo adaptor, as previously suggested. Moreover, we show that HIV-1 motility is supported by multiple, diverse dynein cargo adaptors as HIV-1 binds to dynein light and intermediate chains on dynein's tail. Further, we demonstrate that multiple dynein motors tethered to rigid cargoes, like HIV-1 capsids, display reduced motility, distinct from the behavior of multiple motors on membranous cargoes. Our results introduce a new model of viral trafficking wherein a pathogen opportunistically 'hijacks' the microtubule transport machinery for motility, enabling multiple transport pathways through the host cytoplasm.

Probing the active site chemistry of ß-glucosidases along the hydrolysis reaction pathway.

ß-Glucosidases (EC 3.2.1.21) can be found in all domains of living organisms, where they play essential roles in the removal of terminal glucosyl residues from nonreducing ends of saccharides and glycosides. Two active site amino acid residues, a nucleophile and a proton donor, play key roles in the hydrolytic mechanism. Besides these two highly conserved catalytic residues, there are other conserved amino acids in the active site of ß-glucosidases that make direct hydrogen bonds to the glycosyl moiety at the -1 subsite. In this study, the catalytic mechanism of a GH1 ß-glucosidase (BGlu1) is systematically studied. On the basis of the quantum mechanical studies, the side chain of Tyr315 in an interaction with both O5 of the glucose ring and the nucleophilic glutamate contributes significantly to the energy profile. Glu440 and the conserved Asn175 are the other residues in the polar interaction with -1 glucose with considerable influence on the free energy of the reaction. Gln29, His130, and Trp441, which also form hydrogen bonds to the glycosyl moiety, are found to have relatively a minor effect on the reaction. Different arrangements of active site residues in the high-level [quantum mechanics (QM)] and low-level [molecular mechanics (MM)] regions during the hybrid QM/MM calculations indicate that Tyr315 lowers the energy barrier in the deglycosylation step (by 11.95 kcal/mol) while Glu440 mainly reduces the energy barrier of the glycosylation step. Exclusion of either of these two residues from the QM region results in deviation of the geometric parameters of the enzyme-substrate complex from those expected for the preactivated distorted structure of the substrate.

Study and design of stability in GH5 cellulases.

Thermostable enzymes that hydrolyze lignocellulosic materials provide potential advantages in process configuration and enhancement of production efficiency over their mesophilic counterparts in the bioethanol industry. In this study, the dynamics of ß-1,4-endoglucanases (EC: 3.2.1.4) from family 5 of glycoside hydrolases (GH5) were investigated computationally. The conformational flexibility of 12 GH5 cellulases, ranging from psychrophilic to hyperthermophilic, was investigated by molecular dynamics (MD) simulations at elevated temperatures. The results indicated that the protein flexibility and optimum activity temperatures are appreciably correlated. Intra-protein interactions, packing density and solvent accessible area were further examined in crystal structures to investigate factors that are possibly involved in higher rigidity of thermostable cellulases. The MD simulations and the rules learned from analyses of stabilizing factors were used in design of mutations toward the thermostabilization of cellulase C, one of the GH5 endoglucanases. This enzyme was successfully stabilized both chemically and thermally by introduction of a new disulfide cross-link to its highly mobile 56-amino acid subdomain.

A kinesin-1 variant reveals motor-induced microtubule damage in cells.

Kinesins drive the transport of cellular cargoes as they walk along microtubule tracks; however, recent work has suggested that the physical act of kinesins walking along microtubules can stress the microtubule lattice. Here, we describe a kinesin-1 KIF5C mutant with an increased ability to generate damage sites in the microtubule lattice as compared with the wild-type motor. The expression of the mutant motor in cultured cells resulted in microtubule breakage and fragmentation, suggesting that kinesin-1 variants with increased damage activity would have been selected against during evolution. The increased ability to damage microtubules is not due to the enhanced motility properties of the mutant motor, as the expression of the kinesin-3 motor KIF1A, which has similar single-motor motility properties, also caused increased microtubule pausing, bending, and buckling but not breakage. In cells, motor-induced microtubule breakage could not be prevented by increased α-tubulin K40 acetylation, a post-translational modification known to increase microtubule flexibility. In vitro, lattice damage induced by wild-type KIF5C was repaired by soluble tubulin and resulted in increased rescues and overall microtubule growth, whereas lattice damage induced by the KIF5C mutant resulted in larger repair sites that made the microtubule vulnerable to breakage and fragmentation when under mechanical stress. These results demonstrate that kinesin-1 motility causes defects in and damage to the microtubule lattice in cells. While cells have the capacity to repair lattice damage, conditions that exceed this capacity result in microtubule breakage and fragmentation and may contribute to human disease.

Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

Nerve cells in the human body can reach up to one meter in length. Different regions of a nerve cell require different materials to perform their roles. The motor proteins kinesins and dynein help to transport the required 'cargo', by moving in opposite directions along tracks called microtubules. However, many cargos have both motors attached, resulting in a tug-of-war to determine which direction and how fast the cargo will travel. In many neurodegenerative diseases, including Alzheimer's, this cargo transport goes awry, so a better understanding of exactly how this process works may help to develop new therapies. There are three families of kinesin motors, for a total of about a dozen different kinesins that engage in this process. Motors in each of the three families have different mechanical properties. Specific cargos also tend to have specific kinesins attached to them. Here Gicking et al. hypothesized that when pulling against dynein in a tug-of-war, kinesins from the three families would behave differently. To test this hypothesis, Gicking et al. linked one kinesin to one dynein motor, one at a time in a test tube, and then observed how these two-motor complexes moved using fluorescence microscopy techniques. Unexpectedly, kinesins from the three different families competed similarly against dynein: there were no clear winners and losers. By incorporating previously published data describing the different motor behaviors, Gicking et al. developed a computational model that provided deeper insight into how this mechanical tug-of-war works. The modeling indicated that kinesins from the three families use different approaches for competing against dynein. Kinesin-1 motors tended to pull steadily against dynein, only detaching relatively rarely, but then take some time to attach back to the microtubule track. In contrast, kinesin-3 motors detached easily when they pull against dynein, but they attach back to the microtubule track quickly, taking only about a millisecond to start moving again. Kinesin-2 motors exhibited an intermediate behavior. Overall, these experiments suggest that the mechanical properties of the motor proteins are not the main factors determining the direction and speed of the cargo. In other words, the outcome of this molecular tug-of-war does not necessarily depend on which motor is stronger or faster. Rather, further mechanisms, including regulation of the adapter molecules that connect the motors to their cargo, may help to regulate which cargo go where in branched nerve cells. A better knowledge of how all these different factors work together will be important for understanding how cargo transport in nerve cells is disrupted in neurodegenerative diseases.

Golgi-associated BICD adaptors couple ER membrane penetration and disassembly of a viral cargo.

During entry, viruses must navigate through the host endomembrane system, penetrate cellular membranes, and undergo capsid disassembly to reach an intracellular destination that supports infection. How these events are coordinated is unclear. Here, we reveal an unexpected function of a cellular motor adaptor that coordinates virus membrane penetration and disassembly. Polyomavirus SV40 traffics to the endoplasmic reticulum (ER) and penetrates a virus-induced structure in the ER membrane called "focus" to reach the cytosol, where it disassembles before nuclear entry to promote infection. We now demonstrate that the ER focus is constructed proximal to the Golgi-associated BICD2 and BICDR1 dynein motor adaptors; this juxtaposition enables the adaptors to directly bind to and disassemble SV40 upon arrival to the cytosol. Our findings demonstrate that positioning of the virus membrane penetration site couples two decisive infection events, cytosol arrival and disassembly, and suggest cargo remodeling as a novel function of dynein adaptors.

A salt-bridge controlled by ligand binding modulates the hydrolysis reaction in a GH5 endoglucanase.

Cellulases, distributed in at least 15 families of glycoside hydrolases, will play a key role in biomass conversion and renewable energy challenges of the future. Cel5B from Clostridium thermocellum is a ß-1,4-endoglucanase and a member of family 5 of glycoside hydrolases (GH5) and is characterized by an (α/ß)(8) barrel structure. In contrast to other retaining enzymes, in which the catalytic carboxylate groups (glutamate or aspartate) are positioned ≈ 5.5 Å apart to facilitate nucleophilic attack on the anomeric carbon of the sugar substrate, these two residues in Cel5B are positioned ≈ 10 Å from each other in the unliganded wild-type structure. The structure of the enzyme solved in complex with a cleavage product (cellobiose) revealed ligand-induced conformational changes in the loop carrying Glu140 (proton donor). The reorientation of Glu140 in the complex reduces the separation of the catalytic glutamate residues to 4.3 Å. In this study, we took advantage of conventional and steered molecular dynamics (MD) simulations along with in silico and in vitro mutagenesis to investigate the ligand-induced changes of the enzyme and interactions involved in preservation of Cel5B conformations in the presence and absence of substrate. We determined that the variation in separation of catalytic glutamates in the absence and presence of substrate is due to the different protonation states of the proton donor glutamate that is largely governed by conformational changes in the ß3α3 loop. In the absence of substrate, the conformation of Cel5B is preserved by an electrostatic interaction between deprotonated Glu140 and protonated His91. The ion pair is interrupted upon the binding of substrate, and the positional displacement of the ß3α3 loop allows Glu140 to become oriented within the active site in a less hydrophilic microenvironment that assists in Glu140 protonation.