Bond Cleavage Frequencies Research Articles

Purification and Characterization of a Less Randomly Acting Endo-1,4-β-D-glucanase from the Culture Filtrates of Fusarium oxysporum

An extracellular endo-1,4-β-D-glucanase from Fusarium oxysporum was purified by affinity chromatography and gel filtration. The enzyme purified in this way was homogeneous when judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. The protein corresponded to a molecular mass and pI value of 41.7 kDa and 6.4, respectively. It was optimally active at pH 4.5 and at 55°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and unsubstituted and substituted cello-oligosaccharides but was inactive on Avicel, filter paper, xylan, cellobiose, p-nitrophenyl-β-D-glucoside, and p-nitrophenyl-β-D-xyloside. However, the enzyme effected only a small change in viscosity of CMC per unit increase of reducing sugar. When cellotriose, cellotetraose, and cellopentaose were used as substrates, the enzyme released mainly cellobiose. Use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the glycosidic bond adjacent to 4-methylumbelliferone. Thus, the purified enzyme appeared to be a less randomly acting endoglucanase.

Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85

The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles.

Alteration of the cleavage mode and of the transglycosylation reactions of the xylanase A of Streptomyces lividans 1326 by site-directed mutagenesis of the Asn173 residue.

The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties. A time-course hydrolysis of xylan showed that the altered xylanase ([N173D] Xln A) initially produced larger amounts of xylose (X1), xylobiose (X2) and xylotriose (X3) than Xln A, but less xylotetraose (X4). The bond-cleavage frequencies were determined for both enzymes using xylopentaose (X5), xylotetraose (X4) and xylotriose (X3) labelled at the reducing end of the molecule. Xln A hydrolysed X5, yielding 56% X2 and 44% X3, while [N173D]Xln A liberated 90% X2 and only 10% X3. Both enzymes hydrolysed X4 into 100% X2 and X3 into 100% X1. Transglycosylation reactions were detected in HPLC hydrolysis patterns using high substrate concentrations, where larger products than the starting substrates were formed. Their subsequent degradation also affected the yield of hydrolysis products. Using X5 as substrate, products from xylohexaose (X6) up to xylooligosides larger than xylooctaose (X8) were synthesized by Xln A, while [N173D]Xln A produced only a small amount of xyloheptaose (X7) and X8. Xln A hydrolysed X5 into an equivalent amount of X4 and X2 and 1.5-fold more X3. However, [N173D]Xln A yielded the same amount of X3 and X2 but half as much X4. With X4 as substrate, Xln A synthesized twofold more X7 and X6 than [N173D]Xln A. Xln A liberated 1.4-fold more X3 than X2, while [N173D]Xln A yielded twofold more X2 than X3. Xln A liberated almost fourfold more X2 than X1 from X3, while [N173D]Xln A produced only twofold more X2 than X1. These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reaction catalysed by this xylanase.

Mode of action of three endo-β-1,4-xylanases of Streptomyces lividans

The mode of action of three genetically distinct endo-beta-1,4-xylanases (EXs) of Streptomyces lividans, XlnA, XlnB and XlnC, belonging to two different xylanase families, was investigated on a variety of polysaccharide and oligosaccharide substrates. Viscosimetric measurements showed that all three enzymes have about the same endo-acting character. Occurrence of multiple pathways of substrate degradation at high concentration of beta-1,4-xylooligosaccharides suggested that all three enzymes were retaining glycanases. The enzymes differed considerably in their mode of action on various heteroxylans and on rhodymenan. XlnA hydrolyzed all tested polysaccharides to a higher degree than XlnB or XlnC, through liberation of smaller hydrolysis products, both linear or branched. XlnA performed much better than XlnB or XlnC, particularly on acetylxylan, liberating large amounts of short acetylated and non-acetylated fragments. XlnB and XlnC liberated from acetylxylan only limited amounts of larger acetylated fragments. XlnA exhibited also much higher catalytic efficiency than the other two EXs on short beta-1,4-xylooligosaccharides. The kinetic parameters and bond-cleavage frequencies determined for xylotriose, xylotetraose and xylopentaose using 1-3H-reducing-end-labelled compounds suggested that the substrate binding site of XlnA is smaller and differently organized than those in XlnB or XlnC. In contrast to XlnB and XlnC, XlnA also exhibited significant aryl-beta-xylosidase activity. No distinctive catalytic properties of either XlnB or XlnC were found which were not inherent also to XlnA. High-molecular-mass EXs of the XlnA type show much greater catalytic versatility due than low-molecular-mass EXs of the XlnB or XlnC type.

The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans.

Talaromyces emersonii, a thermophilic aerobic fungus, produces a complete xylan-degrading enzyme system when grown on appropriate substrates. In this paper we present the physicochemical and catalytic properties of three enzymes, xylosidase (Xyl) I (M(r) 181,000; pI 8.9), II (M(r) 131,000; pI 5.3) and III (M(r) 54,200; pI 4.2). Xyl I and II appear to be dimeric and Xyl III is a single-subunit protein. All three enzymes catalyse the hydrolysis of aryl beta-D-xylosides and xylo-oligosaccharides. Xyl I is a classic beta-xylosidase (1,4-beta-D-xylan xylohydrolase; EC 3.2.1.37), and Xyl II and III are novel xylanases (endo-1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) which we believe have not hitherto been reported. In addition to the above substrates, they also catalyse the extensive hydrolysis of unsubstituted xylans, and may have considerable biotechnological potential. The hydrolysis product profiles and bond-cleavage frequencies with various substrates are presented.

Action pattern of xylo‐oligosaccharide hydrolysis by Schizophyllum commune xylanase A

The endo-1,4-beta-xylanase of the basidiomycete Schizophyllum commune, designated xylanase A, was studied to determine its action pattern, rates of reaction and bond-cleavage frequencies on xylo-oligomer and xylo-alditol substrates ranging in degree of polymerization (Dp) from xylotriose (X3) to xyloheptaose (X7). An HPLC method using a Dionex HPLC and Carbopac PA1 ion-exchange column with pulsed amperometric detection was developed to quantify both substrate loss and increase of products. Xylanase A had no detectable activity on xylobiose (X2) and low activity on xylotriose and xylotetraose (X4) but cleaved X5-X7 rapidly with X2 and X3 as major products. Initial rate data from hydrolyses of individual oligomers at 25 degrees C and pH 5.81 indicated that the Michaelis constant (Km) decreased with increasing chain length (n) of oligomer. Turnover number (kcat) increased with chain length up to n = 7 suggesting that the specificity region of xylanase A spans about seven xylose units. Bond-cleavage frequencies obtained from xylanase A hydrolysis of xylo-alditols indicated a strong preference for internal linkages of the xylose chain. The action pattern of xylanase A on reduced substrates suggests that the catalytic site is located assymetrically within the binding cleft of the enzyme.

The endo‐1,4‐β‐glucanase I from Trichoderma reesei

The reaction mechanism of the non-specific endo-1,4-beta-glucanase from Trichoderma reesei QM 9414 (endoglucanase I) was investigated using both reducing-end3H-labelled and universally 14C-labelled cellooligosaccharides, as well as reducing-end3H-labelled xylooligosaccharides. The bond cleavage frequencies of cellooligosaccharides proved to be dependent upon the substrate concentration, especially in the case of cellotriose. In addition to simple hydrolytic cleavage, the enzyme catalyzes reactions along alternative pathways, including transglycosylations leading to products larger than the substrate. Some of these pathways were shown to be reversible. During cellotriose or cellopentaose degradation, substrate resynthesis was demonstrated by incorporation of added radioactive D-glucose or cellobiose. The endoglucanase I is active on xylan and xylooligosaccharides, but less than on soluble cellulose derivatives (e.g. hydroxyethylcellulose) and cellooligosaccharides. The fact that for these different types of substrates the same active site is operative is proven by the ability of the enzyme to utilize cellooligosaccharides and xylooligosaccharides as both glycosyl donors and acceptors. The mixed substrate reactions lead to products composed of D-glucosyl and D-xylosyl residues. The kinetic parameters for cellooligosaccharide degradation can be used for the description of an extended substrate binding site. Of the four putative glycosyl subsites, -II and +II show the highest affinities, 16.7 kJ.mol-1 and 7.1 kJ.mol-1, respectively.

Kinetics and subsite mapping of a d-xylobiose- and d-xylose-producing Aspergillus niger endo-(1→4)-β- d-xylanase

A previously described endo-(1→4)-β- d-xylanase produced by Aspergillus niger was allowed to react with linear unlabeled and labeled d-xylo-oligosaccharides ranging from d-xylotriose to d-xylo-octaose. No evidence of multiple attack or of condensation and trans- d-xylosylation reactions was found. Maximum rates and Michaelis constants were measured at 40° and pH 4.85. The former increased with increasing chain-length from d-xylotriose through d-xylohexaose to ∼70% of that on soluble larchwood d-xylan, and then decreased slightly for d-xyloheptaose and d-xylo-octaose. Michaelis constants decreased monotonically with increasing chain-length. Bond-cleavage frequencies were highest near the reducing end of short substrates, with the locus of highest frequencies moving towards the middle of larger substrates. These data indicated that the endo- d-xylanase has five main subsites, with the catalytic site located between the third and fourth subsites, counting from the nonreducing end of the bound substrate. The subsite to the nonreducing side of the catalytic site strongly repels its corresponding d-xylosyl residue, while the two subsites farther towards the nonreducing end of the substrate strongly attract their corresponding residues. The subsite to the reducing site of the catalytic site moderately attracts d-xylosyl residues, while the next one towards the reducing end has a high affinity for them. The residual error of the numerical estimation was allocated largely to the Michaelis constants of the different d-xylo-oligosaccharides, whose calculated values were appreciably smaller than measured values, especially for shorter substrates. This suggests that the subsite model cannot fully account for the experimental data. Estimated and measured values of maximum rates, bond-cleavage frequencies, and dissociation constant when the active site is fully occupied by substrate agreed more closely with each other.

The determination of subsite binding energies of porcine pancreatic α-amylase by comparing hydrolytic activity towards substrates

The active centre of porcine pancreatic α-amylase contains five subsites. Their occupancy has been studied using as a substrate maltooligosaccharide of various chain lengths (maltose up to maltoheptaose), some of their p- and o-nitrophenylated derivatives, and 412-residue amylose. Quantitative analysis of the digestion products allowed the determination of the subsite occupancy for the various productive complexes, the bond cleavage frequency and respective k cat i (where i is the binding mode). The catalytic efficiency ( k cat/ K m) increases with chain length from maltose (2 M −1·s −1) up to amylose (1.06·10 7 M −1·s −1). The kinetic parameters of p-nitrophenylmaltoside hydrolysis are quite close to those of maltose, and the ortho compound behaves as maltotriose. Determination of binding energy of glucose residue at the various subsites calculated according to the method of Hiromi et al. (Hiromi, K., Nitta Y. Numata, C. and Ono, S. (1973) Biochim. Biophys. Acta 302, 362–375) did not give consistent results. A method is proposed based on certain properties of porcine pancreatic α-amylase, especially the non-interaction of the p-nitrophenyl moiety of the maltose derivative with subsites 1 and 2, and the o-nitrophenyl group which interacts in a similar way to a glucose residue at the reducing end, and on the grounds that the amylase-amylose complexes are of the productive type. In addition, binding energy differences were calculated from substrates with the same chain length. The subsite energy profile is characterized by a low value at subsite 3 which confirms this subsite as the catalytic one. Another consequence is that the hydrolysis rate constant of productive complexes ( k int n ) (where n is the number of glucose or glucose equivalent residues for a given substrate) varies with chain length which is in conflict with the hypothesis of Hiromi et al.

A study of the pattern of action of endo-(1→3)-β- d-glucanases from marine bivalves on a polymer substrate labelled at the reducing end

The pattern of action of L-IV and L-O endo-laminarinases (EC 3.2.1.6) from the crystalline style of the marine bivalves Spisula sachalinensis and Chlamys albidus, respectively, on Smith-degraded laminarin labelled with 3H at the reducing end has been studied. The average d.p. of the substrate was 25. On the basis of relative bond-cleavage frequencies. The L-IV enzyme was shown to attack the substrate molecule more than once per encounter. The degree of this repetitive attack and the subsite affinities of the aglycon part of the active centre of both (1→3)-β- d-glucanases have been evaluated.

The active site of an acidic endo-1,4-β-xylanase of Aspergillus niger

The substrate binding site of an acidic endo-1,4-β-xylanase (1,4-β- d-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-β-xylooligosaccharides (1- 3H)-labelled at the reducing end. Bond cleavage frequencies and V/ K m parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293–316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xyiosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger β-xylanase appears to be an endo-1,4-β-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate.

Mechanisms of substrate digestion by endo-1,4-beta-xylanase of Cryptococcus albidus. Lysozyme-type pattern of action.

The action pattern and reaction mechanism of the endo-1,4-beta-xylanase of the yeast Cryptococcus albidus were investigated using reducing-end (1-3H)-labelled and uniformly 14C-labelled beta-1,4-xylooligosaccharides up to xylopentaose. The enzyme was found to catalyze degradation of oligosaccharides also by other pathways than a simple hydrolytic cleavage. Bond-cleavage frequency of xylotriose, xylotetraose and xylopentaose were found to be concentration dependent. At high substrate concentration reactions such as xylosyl, xylobiosyl and xylotriosyl transfer occur and result in the formation of products larger than the starting substrate. Xylose and xylobiose to significant extent enter the reaction pathways as glycosyl acceptors. None of the transglycosylic reactions observed with reducing-end-labelled substrates or acceptors were accompanied by a significant label redistribution from the reducing-end unit, suggesting that the enzyme-glycosyl intermediates effective in the transfer reactions can be formed from the non-reducing-end units of oligosaccharides. Evidence for the formation of a termomolecular shifted complex of beta-xylanase with xylotriose has also been obtained. All features of the degradation of oligosaccharides by beta-xylanase are consistent with the lysozyme-type reaction mechanism.

10] Subsite mapping of enzymes: Application to polysaccharide depolymerases

This chapter discusses subsite mapping, the term applied to the experimental determination of the number of subsites, comprising the binding region, the energetics of interaction of each subsite with a monomer residue, and the hydrolytic rate coefficients. The value of the generated subsite map lies in its ability to correctly predict the action pattern of the enzyme. This development is restricted to subsite mapping of polysaccharide depolymerases; however, the general principles developed here are also applicable, within certain limitations, to other polymer-acting enzymes. The interactions of maltotetraose with the binding region of a hypothetical five-subsite amylase are discussed in the chapter. If the substrate binds to expose a susceptible bond to the catalytic amino acids on the enzyme, the complex is productive and bond cleavage can occur. The remaining positional isomers are nonproductive in the sense that they cannot lead to bond scission. The rates of bond scission of the substrate depend on the energetic of binding of the productive positional isomers and on the respective hydrolytic rate coefficients. The chapter explains the best overall procedure for mapping of the binding region of a polysaccharide depolymerase. These are as follows: (1) test for complicating reactions, (2) use bond cleavage frequencies to establish the binding region topography and to measure the apparent subsite binding energies, and (3) use Km and V measured as a function of substrate chain length to complete the subsite map.

Subsite mapping of enzymes. Application of the depolymerase computer model to two alpha-amylases.

In the preceding paper (Allen and Thoma, 1976) we developed a depolymerase computer model, which uses a minimization routine to establish a subsite map for a depolymerase. In the present paper we show how the model is applied to experimental data for two alpha-amylases. Michaelis parameters and bond-cleavage frequencies for substrates of chain lengths up to twelve glucosyl units have been reported for Bacillus amyloliquefaciens, and a subsite map has been proposed for this enzyme [Thoma et al. (1971) J. Biol. Chem. 246, 5621-5635]. By applying the computer model to the experimental data, we have arrived at a ten-subsite map. We find that a significant improvement in this map is achieved by allowing the hydrolytic rate coefficient to vary as a function of the number of occupied subsites comprising the enzyme-binding region. The bond-cleavage frequencies, the enzyme is found to have eight subsites. A partial subsite map is arrived at, but the entire binding region cannot be mapped because Michaelis parameters are complicated by transglycosylation reactions. The hydrolytic rate coefficients for this enzyme are not constant.

Subsite mapping of enzymes. Depolymerase computer modelling

We have developed a depolymerase computer model that uses a minimization routine. The model is designed so that, given experimental bond-cleavage frequencies for oligomeric substrates and experimental Michaelis parameters as a function of substrate chain length, the optimum subsite map is generated. The minimized sum of the weighted-squared residuals of the experimental and calculated data is used as a criterion of the goodness-of-fit for the optimized subsite map. The application of the minimization procedure to subsite mapping is explored through the use of simulated data. A procedure is developed whereby the minimization model can be used to determine the number of subsites in the enzymic binding region and to locate the position of the catalytic amino acids among these subsites. The degree of propagation of experimental variance into the subsite-binding energies is estimated. The question of whether hydrolytic rate coefficients are constant or a function of the number of filled subsites is examined.