Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product.

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

Analysis of Factors Related to Variation in Dissolution Profiles Estimated from Continuously Conducted Dissolution Tests of Generic Products.

The development of generic pharmaceuticals involves a bioequivalence study to ensure the therapeutic equivalence of the test formulation to the original innovative product. The formulation characteristics of generic products are expected to be maintained in the long term after approval. This study analyzed the factors contributing to the changes in the dissolution profiles of approved products during their life cycles. Cumulative data on the dissolution similarity of 1675 products of 127 ingredients tested by official laboratories in Japan were assessed according to Japanese bioequivalence guidelines with slight modifications. The products showing dissimilarities in dissolution profiles were analyzed for reporting year, therapeutic category, co-development, physical properties of the active pharmaceutical ingredient (API), and suspected reasons for dissolution change. The increase in the number of dissimilar products is related to the co-development of generic products. Although the solubility of the API was not associated with the dissolution change in the analysis of the total dissolution data, control of the API particle size is suggested to be important for drugs with poorly soluble APIs. Additionally, a risk factor for dissolution changes in the test solutions at a certain pH was the presence of acidic or basic residues. These results indicate the importance of proper development through a thorough evaluation of the formulation and process factors affecting the dissolution properties throughout the product lifecycle.

Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Rationale, design and baseline characteristics of the effect of canagliflozin in patients with type 2 diabetes and microalbuminuria in the Japanese population: The CANPIONE study.

AIM: To evaluate the effect of canagliflozin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, on albuminuria and the decline of estimated glomerular filtration rate (eGFR) in participants with type 2 diabetes and microalbuminuria. METHODS: The CANPIONE study is a multicentre, randomized, parallel-group and open-labelled study consisting of a unique 24-week preintervention period, during which the rate of eGFR decline before intervention is estimated, followed by a 52-week intervention and a 4-week washout period. Participants with a geometric mean urinary albumin-to-creatinine ratio (UACR) of 50 and higher and less than 300 mg/g in two consecutive first-morning voids at two different time points, and an eGFR of 45 ml/min/1.73m2 or higher, are randomly assigned to receive canagliflozin 100 mg daily or to continue guideline-recommended treatment, except for SGLT2 inhibitors. The first primary outcome is the change in UACR, and the second primary outcome is the change in eGFR slope. RESULTS: A total of 258 participants were screened and 98 were randomized at 21 sites in Japan from August 2018 to May 2021. The mean baseline age was 61.4 years and 25.8% were female. The mean HbA1c was 7.9%, mean eGFR was 74.1 ml/min/1.73m2 and median UACR was 104.2 mg/g. CONCLUSIONS: The CANPIONE study will determine whether the SGLT2 inhibitor canagliflozin can reduce albuminuria and slow eGFR decline in participants with type 2 diabetes and microalbuminuria.

Head Injury Assessment in the Elite Level Rugby Union in Japan: Review of 3 Seasons.

Head Injury Assessment (HIA) is the screening tool for head injury during a rugby game. The purpose of this study was to investigate the epidemiology of HIA in the Japan Rugby Top League (JRTL). The incidences of HIA, defined concussion (per 1,000 player-hours) and repeated concussions were evaluated in three seasons (2016-17, 2017-18, 2018-19; total 360 games). The HIA incidence rates were 12.7 (95% confidence interval 9.5-15.9), 20.8 (16.8-24.9), and 25.0 (20.5-29.5) in each season. HIA-1 criteria 2, which is applied for suspected concussion cases, was performed for 46 cases in the 2016-17 season, 81 cases in the 2017-18 season, and 88 cases in the 2018-19 season. The concussion incidence rates were significantly greater in the 2017-18 season (9.6/1000 player-hours, 95% confidence interval 6.8-12.4) and the 2018-19 season (14.4, 11-17.8) compared to the 2016-17 season (4.8, 2.8-6.8). The number of repeated concussion cases in the same season was 1 in the 2016-17 season and 4 in both the 2017-18 and 2018-19 seasons. This study confirmed significantly higher HIA and concussion incidence rates over time. Although the HIA system might have been established in the three seasons in JRTL, comprehensive management needs to be improved to prevent repeated concussions.

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens.

Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.

HEATR1, a novel interactor of Pontin/Reptin, stabilizes Pontin/Reptin and promotes cell proliferation of oral squamous cell carcinoma.

Pontin and Reptin are closely related proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They form a hetero-oligomeric complex, Pontin/Reptin, which is involved in protein stability and assembly of the protein complexes as a molecular chaperone. Overexpression of Pontin and Reptin in tumor cells has been reported and is implicated in the development of various cancers. However, the molecular mechanism of Pontin/Reptin function in oral squamous cell carcinoma (OSCC) development remains unclear. Here, we identify HEAT repeat-containing protein 1 (HEATR1) as a novel binding factor of Pontin/Reptin. Functionally, HEATR1 stabilizes Pontin/Reptin and positively regulates OSCC cell proliferation by activating mTOR and pre-rRNA synthesis. We also find that HEATR1 expression is markedly upregulated in tumor region of OSCC tissue. Hence, we propose that HEATR1 is involved in the regulation of mTOR and ribosome biogenesis as a potential protein stabilizer of Pontin/Reptin in OSCC.

Label-free monitoring of crystalline chitin hydrolysis by chitinase based on Raman spectroscopy.

We demonstrate a method for label-free monitoring of hydrolytic activity of crystalline-chitin-degrading enzyme, chitinase, by means of Raman spectroscopy. We found that crystalline chitin exhibited a characteristic Raman peak at 2995 cm-1, which did not appear in the reaction product, N,N'-diacetylchitobiose. We used this Raman peak as a marker of crystalline chitin degradation to monitor the hydrolytic activity of chitinase. When the crystalline chitin suspension and chitinase were mixed together, the peak intensity of crystalline chitin at 2995 cm-1 was linearly decreased depending on incubation time. The decrease in peak intensity was inversely correlated with the increase in the amount of released N,N'-diacetylchitobiose, which was measured by conventional colorimetric assay with alkaline ferricyanide. Our result, presented here, provides a new method for simple, in situ, and label-free monitoring of enzymatic activity of chitinase.

Increased CD69 expression on activated eosinophils in eosinophilic chronic rhinosinusitis correlates with clinical findings.

BACKGROUND: Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis associated with asthma. CD69 is an important marker of activation for eosinophils. But, whether a correlation exist between the CD69 expression on eosinophils and clinical findings is unclear. METHODS: We performed quantitative PCR and/or flow cytometry using tissue and purified eosinophils from the blood and nasal polyps of 12 patients with ECRS and from 8 patients without ECRS (controls). We assessed clinical findings including nasal polyp (NP) scores, sinus CT findings, and pulmonary function test results, and examined their possible association with the CD69 expression. We also performed CD69 cross-linking experiments in mouse eosinophils to investigate the functional role of CD69. RESULTS: Levels of cytokine mRNAs (IL-4, -5, -10, and -13) were significantly higher in purified NP eosinophils and tissues from patients with ECRS than the levels of those in controls. The expressions of major basic protein (MBP), eosinophilic cationic protein (ECP), eosinophilic-derived neurotoxin (EDN), eosinophil peroxidase (EPX) in cytotoxic granules, and CD69 mRNA were significantly higher in purified eosinophils from NPs than in those from blood. We also found a correlation between expression of CD69 and clinical findings. Moreover, we found EPX release from mouse eosinophils following CD69 cross-linking. CONCLUSIONS: These data suggest that increased CD69 expression by eosinophils is not only a biomarker for nasal obstruction and pulmonary dysfunction, but also a potential therapeutic target for patients with ECRS and asthma.

Spectator medicine at an international mega sports event: Rugby World Cup 2019 in Japan.

BACKGROUND: The Rugby World Cup (RWC) is one of the biggest international mega sports events in the world. This study was conducted to identify and evaluate the volume, nature, and severity of spectator medical care in the stadiums of 12 venues across Japan during RWC 2019. METHOD: This was a retrospective review of medical records from spectator medical rooms of 45 official matches of RWC 2019 between September 20 and November 2, 2019. All patients in the stadium who visited the spectator medical room and were transferred to a hospital were included. The wet bulb globe temperature (WBGT) value at the kick-off time of each match, the number of visits to the spectator medical room, and the number of transfers to a hospital were reviewed and analyzed. The patient presentation rate (PPR) was calculated per 10,000 attendees. Severity categories were defined as mild or severe. Mild cases were considered non-life threatening requiring minimal medical intervention, and severe cases required transport to a hospital. RESULT: The total number of visits to the spectator medical room was 449 with a PPR of 2.63. Most cases (91.5%) were mild in severity. The PPR was significantly higher for the matches held with a WBGT over 25 °C than for the matches under 21 °C (PPR 4.27 vs 2.04, p = 0.04). Thirty-eight cases were transferred to a hospital by ambulance; the PPR was 0.22. The most common reasons for transfer to the hospital were heat illness and fracture/dislocation, at a rate of 15.8% each. The incidence rate of cardiopulmonary arrest per 10,000 attendees was 0.0059 during RWC 2019. CONCLUSION: Preparation and provision of appropriate medical service for spectators is a key factor for mass-gathering events. During RWC 2019, the majority (91.5%) of patients who sought medical attention did so for minor complaints, which were easily assessed and managed. On the other hand, a higher WBGT situation contributes significantly to an increased PPR (< 21 versus > 25, 2.04 versus 4.27, p = 0.04). Careful medical preparation, management, and development of public education programs for higher WBGT situations will be required in the future for similar international mega sports events.

Single-Nanoparticle Tracking with Angstrom Localization Precision and Microsecond Time Resolution.

Gold nanoparticles (AuNPs) have been used as a contrast agent for optical imaging of various single biomolecules. Because AuNPs have high scattering efficiency without photobleaching, biomolecular dynamics have been observed with nanometer localization precision and sub-millisecond time resolution. To understand the working principle of biomolecular motors in greater detail, further improvement of the localization precision and time resolution is necessary. Here, we investigated the lower limit of localization precision achievable with AuNPs and the fundamental law, which determines the localization precision. We first used objective-lens-type total internal reflection dark-field microscopy to obtain a scattering signal from an isolated AuNP. The localization precision was inversely proportional to the square root of the photon number, which is consistent with theoretical estimation. The lower limit of precision for a 40 nm AuNP was ∼0.3 nm with 1 ms time resolution and was restricted by detector saturation. To achieve higher localization precision, we designed and constructed an annular illumination total internal reflection dark-field microscopy system with an axicon lens, which can illuminate the AuNPs at high laser intensity without damaging the objective lens. In addition, we used high image magnification to avoid detector saturation. Consequently, we achieved 1.3 Å localization precision for 40 nm AuNPs and 1.9 Å localization precision for 30 nm AuNPs at 1 ms time resolution. Furthermore, even at 33 µs time resolution, localization precisions at 5.4 Å for 40 nm AuNPs and at 1.7 nm for 30 nm AuNPs were achieved. We then observed motion of head of kinesin-1 labeled with AuNP at microsecond time resolution. Transition cycles of bound/unbound states and tethered diffusion of unbound head during stepping motion on microtubule were clearly captured with higher time resolution or smaller AuNP than those used in previous studies, indicating applicability to single-molecule imaging of biomolecular motors.

Single-molecule imaging and manipulation of biomolecular machines and systems.

BACKGROUND: Biological molecular machines support various activities and behaviors of cells, such as energy production, signal transduction, growth, differentiation, and migration. SCOPE OF REVIEW: We provide an overview of single-molecule imaging methods involving both small and large probes used to monitor the dynamic motions of molecular machines in vitro (purified proteins) and in living cells, and single-molecule manipulation methods used to measure the forces, mechanical properties and responses of biomolecules. We also introduce several examples of single-molecule analysis, focusing primarily on motor proteins and signal transduction systems. MAJOR CONCLUSIONS: Single-molecule analysis is a powerful approach to unveil the operational mechanisms both of individual molecular machines and of systems consisting of many molecular machines. GENERAL SIGNIFICANCE: Quantitative, high-resolution single-molecule analyses of biomolecular systems at the various hierarchies of life will help to answer our fundamental question: "What is life?" This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

Correction: Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem. Chem. Phys., 2018, DOI: .

Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis.

Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 µm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M µm and 8.1 × 10-10 M µm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

Visualization of Functional Structure and Kinetic Dynamics of Cellulases.

Cellulose is the most abundant carbohydrate on earth and hydrolyzed by cellulases in nature. During catalysis, cellulase transfers protons to and from the oxygen atoms of the glycosidic bond and a water molecule. Since cellulose is an insoluble polymer, some kinds of cellulases, with high activity toward crystalline cellulose, move on the crystal surface with continuous hydrolysis of the molecular chain. In addition, binding and dissociation on/from the crystal surface are also important elementary steps of the reaction cycle. Recently, these interesting features of cellulases can be directly analyzed, due to the development of visualization techniques. In this chapter, we introduce (1) visualization of the protonation state of the catalytic residue by neutron crystallography, (2) visualization of processive movement on the crystal surface by high-speed atomic force microscopy , and (3) visualization of binding and dissociation events by single-molecule fluorescence microscopy.

Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose.

Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Iα The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.

Structural and biochemical analyses of glycoside hydrolase family 26 ß-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of ß-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose Iα and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose Iα and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.

The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose.

BACKGROUND: Mutation of Trp-40 in the Cel7A cellobiohydrolase from Trichoderma reesei (TrCel7A) causes a loss of crystalline cellulose-degrading ability. RESULTS: Mutant W40A showed reduced specific activity for crystalline cellulose and diffused the cellulose chain from the entrance of the active site tunnel. CONCLUSION: Trp-40 is essential for chain end loading to initiate processive hydrolysis of TrCel7A. SIGNIFICANCE: The mechanisms of crystalline polysaccharide degradation are clarified. The glycoside hydrolase family 7 cellobiohydrolase Cel7A from Trichoderma reesei is one of the best studied cellulases with the ability to degrade highly crystalline cellulose. The catalytic domain and the cellulose-binding domain (CBD) are both necessary for full activity on crystalline substrates. Our previous high-speed atomic force microscopy studies showed that mutation of Trp-40 at the entrance of the catalytic tunnel drastically decreases the ability to degrade crystalline cellulose. Here, we examined the activities of the WT enzyme and mutant W40A (with and without the CBD) for various substrates. Evaluation and comparison of the specific activities of the enzymes (WT, W40A, and the corresponding catalytic subunits (WTcat and W40Acat)) adsorbed on crystalline cellulose indicated that Trp-40 is involved in recruiting individual substrate chains into the active site tunnel to initiate processive hydrolysis. This was supported by molecular dynamics simulation study, i.e. the reducing end glucose unit was effectively loaded into the active site of WTcat, but not into that of W40Acat, when the simulation was started from subsite -7. However, when similar simulations were carried out starting from subsite -5, both enzymes held the substrate for 50 ns, indicating that the major difference between WTcat and W40Acat is the length of the free chain end of the substrate required to allow initiation of processive movements; this also reflects the difference between crystalline and amorphous celluloses. The CBD is important for enhancing the enzyme population on crystalline substrate, but it also decreases the specific activity of the adsorbed enzyme, possibly by attaching the enzyme to non-optimal places on the cellulose surface and/or hindering processive hydrolysis.