Michael L. Sinnott

Engineering the Exo-loop of Trichoderma reesei Cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

Key Residues in Subsite F Play a Critical Role in the Activity of Pseudomonas fluorescens Subspecies cellulosa Xylanase A Against Xylooligosaccharides but Not Against Highly Polymeric Substrates such as Xylan

In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action. However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F. Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-β1,4-xylanase, providing support for the existence of subsite F. The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft. To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated. N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose. These data suggest that N182 is located at the C site of the enzyme. N126A and K47A were less active against xylan and aryl-β-glycosides than native XYLA. The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed. E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose. These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.

Substrate specificity in glycoside hydrolase family 10: Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of pseudomonas cellulosa xylanase 10A

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes β1,4-linked xylans but exhibits very low activity against aryl-β-cellobiosides. The family 10 enzyme, Cex, fromCellulomonas fimi, hydrolyzes aryl-β-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the –1 and –2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998)Biochemistry 37, 4751–4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the –2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the –1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-β-cellobioside and 4-nitrophenyl-β-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-β-xylobioside activity but did not significantly reduce 4-nitrophenyl-β-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.

Stereochemical course of the action of the cellobioside hydrolases I and II of Trichoderma reesei

With β-cellobiosyl fluoride as substrate, CBHI gives β-cellobiopyranose as the first product, whereas CBHI gives α-cellobiopyranose (CBH = cellobioside hydrolase).

Catalysis by β-glucosidase A3 of Aspergillus wentii

(1)α-Deuterium kinetic isotope effects on Vmax. for hydrolysis of both β-D-glucopyranosylpyridinium ions and aryl β-D-glucopyranosides are in the range kH/kD 1.08–1.14, indicating that bond-breaking limits the rate of hydrolysis of both sets of substrates. The variation of kcat with aglycone acidity, expressed by log kcat=A+βlgpKa, is governed by a βlg value of –0.96 ± 0.19 for the N-glycosides and –0.05 ± 0.05 for the O-glycosides. The latter, low value of |βlg| is evidence for extensive proton donation to the oxygen at the transition state, even for the departure of acidic aglycones. The dependence of rate on protonation of a group of pKa < 6 required by this idea is indeed observed. (2) Comparison of log kcat values with spontaneous hydrolysis rates indicates that nucleophilic and non-covalent interactions accelerate C–N bond cleavage in the ES complex by a factor of 10(8 + 0.3[pka]N) for glucosyl pyridinium salts where [pKa]N is the pKa of the pyridine. On the assumption that these interactions are similar for O-glucosides, proton donation to the aglycone oxygen atom can be estimated to contribute a rate-enhancement of 10(0.1 + 0.7[pKa]O), where [pKa]O is that of the free phenol. (3)D-Glucono-δ-lactone and 5-amino-5-deoxy-D-gluconolactam are bound 102.8 and 102.2 times, respectively, more tightly than β-D-glucopyranose, because of their analogy to a transition state in which α-deuterium kinetic isotope effects have shown the anomeric carbon atom to have substantial sp2 character. (4) Cationic inhibitors are bound 102.5–103.5 times more tightly than directly comparable neutral ones, if the positive charge resides on the equatorial substituent at C-1, but protonated 2-amino-2-deoxyglucose is bound no more tightly than glucose. This indicates the presence of a negative charge in the El complex near C-1 on the α face of the pyranose ring (an aspartate residue previously identified by covalent labelling) which is partly compensated by a positive charge near C-2.

18 O and secondary 2H kinetic isotope effects confirm the existence of two pathways for acid-catalysed hydrolyses of α-arabinofuranosides

The 18O kinetic isotope effect on the HClO4-catalysed hydrolysis of 4-nitrophenyl [1-18O]-α-arabinofuranoside (k16/k18) is 1.023 ± 0.003 at 80.0 °C; that for isopropyl (1-18O]-α-arabinofuranoside is 0.988 at 30.2 °C and the secondary deuterium effect on the hydrolysis of [2-2H]propan-2-yl α-arabinofuranoside (kH/kD) is 0.979. The nitrophenyl glycoside reacts with exocyclic C–O cleavage and the propan-2-yl glycoside by endocyclic C–O cleavage.

Failure of the antiperiplanar lone pair hypothesis in glycoside hydrolysis. Synthesis, conformation, and hydrolysis of α-D-xylopyranosyl-and α-D-glucopyranosyl-pyridinium salts

(1) Two series of crystalline α-D-xylopyranosyl-and α-D-glucopyranosyl-pyridinium bromides have been made, by reaction of the acetylated α-bromides with the pyridines in an aprotic solvent in the presence of bromide ion, followed by deacetylation with aqueous HBr at room temperature. (2) 200 MHz 1H N.m.r. spectra in D2O of representatives of each series show the xylo-compounds to adopt the 1C4 conformation, and the gluco-compounds the 1S3 conformation. (3) The hydrolyses of α-D-xylopyranosyl- and α-D-glucopyranosyl-3-bromopyridinium ions in 1.0M-NaCIO4 at 25.0 °C are described by kobs/s–1=4.7 × 10–8+1.4 × 10–18/[H+] and kobs/s–1=2 × 10–7+1.4 × 10–17/[H+], respectively. (4) The products of pH-independent hydrolysis of the foregoing ions are the sugars and 3-bromopyridine, whereas at pH 12, 10% of both reactions proceed by attack on the pyridine ring: attack on the sugar yields glucose and 1,6-anhydroglucopyranose, but no xylose. (5) The pH-independent hydrolyses display strongly positive entropies of activation: at 25.0 °C, those of five xylo-compounds give a βlg value of –1.27 ± 0.06, and those of four gluco-compounds one of –1.06 ± 0.12. (6) Arguments are presented, in the light of the low α:β rate ratios for both xylo and gluco pyridinium salts (8–23 and 80, respectively, at 25 °C), that departure of a pyridine from C(1) of an aldopyranose ring does not require a conformation in which the leaving group is antiperiplanar to a lone pair of electrons on O(5). (7) The antiperiplanar lone pair hypothesis is shown to be a special case of the principle of least nuclear motion, which is known not to apply to reactions with very product-(or reactant-) like transition states.

Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, kcat/Km, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside.

Effect of aryl substituents on the kinetics of inactivation of glycosidases by glycosylmethylaryltriazenes: examination of the ‘suicide’ nature of these inactivations

The inactivation of the Mg2+-free form of the lacZ-β-galactosidase of Escherichia coli at 25.0 °C by various β-D-galactopyranosylmethylaryltriazenes resembles the spontaneous, rather than the acid-catalysed decomposition of alkylaryltriazenes in that both the maximum first-order rate constant, and the second-order rate constant, are governed by a negative β1g value at pH 7.0 and at pH 8.0. Less extensive data with the β-xylosidase of Penicillium wortmanni and β-D-xylopyranosylmethylaryltriazenes give a similar result. Although the decomposition of the 2-(β-D-galactopyranosyl) ethyl compounds in aqueous solution is 5–10 fold faster than their lower homologues, β-galactosidase inactivation is 3–13 times slower. β-D-Galactopyranosylmethyl-p-nitrophenyltriazene does not inactivate the lectin, RCA ricin.

A temperature-dependent change in the mechanism of acid catalysis of the hydrolysis of p-nitrophenyl β-D-glucopyranoside indicated by oxygen-18 and solvent deuterium kinetic isotope effects

The 18O kinetic isotope effect for hydrolysis of p-nitrophenyl [1-18O]-β-glucopyranoside in 2.0M-HCl, measured by the isotopic quasi-racemate method, is 1.0257 at 65.5 °C and 1.023 at 75.1 °C; there is a literature value of 1.0355 ± 0.0015 at 50.0 °C, measured mass spectrometrically. That this apparent discrepancy arises from a change in the mode of acid catalysis as the temperature is lowered is shown by the strongly temperature-dependent solvent deuterium isotope effect [log (kD2O/kH2O)= 1.84 – 0.595 × 103/T], and by the greater effect of trifluoroacetate buffers at 45.0 than at 85.0 °C: a rough catalytic constant for general acid catalysis by trifluoroacetic acid of ca. 10–7.5 I mol–1 s–1 can be estimated at the former temperature.