Catalytically important amino acid residues in endoglucanases from a mutant strain <Emphasis Type="Italic">Trichoderma</Emphasis> sp. M<Subscript>7</Subscript>

Two endoglucanases, EG-III (49.7 kD) and EG-IV (47.5 kD), from a mutant strain Trichoderma sp. M₇ were modified with several specific reagents. Water-soluble carbodiimide completely inactivated only one of the purified endoglucanases and kinetic analysis indicated that at least two molecules of carbodiimide bind to EG-IV for inactivation. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 3.57·10^?5 mM^?1·min^?1. Both endoglucanases were inhibited by iodoacetamide, but the absence of substrate protection excluded direct involvement of cysteine residues in the catalysis N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate, since the presence of substrates or analogs prior to NBS modification protected the enzymes against inactivation.     

内切葡聚糖苷水解酶的分离纯化和内源荧光性质

利用离子交换和分子筛层析技术从拟康氏木霉Ｓ－３８固态发酵液中提纯了两个内切葡聚糖苷酶组分．测定了一个组分的内源荧光性质,结果表明：该酶分子的内源荧光几乎都来自色氨酸．Ｎ－溴代琥珀酰亚胺（ＮＢＳ）修饰导致了酶活力的完全丧失,但是酶荧光残留了２５％,抑制剂纤维二糖与酶结合可使部分酶荧光得到保护,同时这种结合也可以保护一定的色氨酸荧光不被外来淬灭剂淬灭．     

Role of tryptophan in the spectral and catalytic properties of the copper enzyme, galactose oxidase.

Previous results indicate that a tryptophan residue(s) may interact with the sugar substrate and Cu(II) atom of galactose oxidase (Ettinger, M. J., and Kosman, D. J. (1974), Biochemistry 13, 1248). We now show that N-bromosuccinimide (NBS) reduces enzymatic activity to 2% as two tryptophans are oxidized; only four residues are easily oxidized in the holoenzyme. An enzymatic activity vs. number of residues oxidized profile suggests that this inactivation is probably associated with only one of the first 2 residues oxidized. There is no evidence for chain cleavage or modification of amino acids other than tryptophan. While substrate protection is not afforded by the sugar substrate, the activity-related tryptophan is placed within the active-site locus by spectral evidence. NBS oxidation of two tryptophans results in a marked diminution of the large copper optical-activity transition at 314 nm. Under some reaction conditions, a doubling of ellipticity in the 600-nm region of copper CD is also observed. The effects of the NBS oxidation on the CD spectra of galactose oxidase permit the assignment of the 314-nm CD band to a charge-transfer transition and the 229-nm extremum to a specific tryptophan contribution. The AZZ parameter from electron spin resonance spectra is also markedly reduced by the NBS oxidation. Moreover, while cyanide binds to the native enzyme without reducing the Cu(II) atom, cyanide rapidly reduces the Cu(II) atom to Cu(I) in the NBS-oxidized enzyme. These CD and ESR results are taken to suggest that one aspect of the inactivation by NBS oxidation may be a conversion of the pseudosquare planar copper complex in the native enzyme to a more distorted, towards tetrahedral, complex in the inactivated enzyme. Since the inactivation can be accomplished without affecting binding of the sugar substrate, tryptophan oxidation must affect catalysis per se.     

Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide

S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.     

Possible functional roles of carboxyl and histidine residues in a soluble thiamine-binding protein of Saccharomyces cerevisiae

The reaction of a soluble thiamine-binding protein of Saccharomyces cerevisiae with water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, at pH 4.5, results in a remarkable loss of its binding activity with thiamine. Thiamine above 0.1 mM substantially protects the protein against this inactivation. In addition to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the thiamine-binding protein is also inactivated by diethylpyrocarbonate. The inactivation is time-dependent and follows second-order kinetics. Restoration of the binding activity by incubation of inactivated protein with hydroxylamine was observed. thiamine and pyrithiamine are effective to prevent the inactivation. From these results it is strongly suggested that both the carboxyl and the histidine residues in the protein are involved in the binding site for thiamine. It is proposed that the binding involves interactions between charged groups on the protein with the quaternary nitrogen of the thiazolium moiety and with the basic ring nitrogen of the pyrimidine moiety in thiamine molecule.     

Inactivation of bovine thrombin by water-soluble carbodiimides: the essential carboxyl group has a pKa of 5.51

Bovine thrombin is rapidly and completely (greater than 99%) inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a pseudo-first-order process. A plot of the pseudo-first-order rate constant for inactivation by 20 mM EDC at different pH values from pH 4.0 to 7.7 at 25 degrees C shows that inactivation is critically dependent on the protonated form of an acidic side chain with a pKa of 5.51. Significant protection against inactivation is provided by the competitive inhibitor dansyl-L-arginine N-(3-ethyl-1,5-pentanediyl)amide, suggesting that the essential carboxyl group may be involved in substrate binding. 1-Ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) inactivates thrombin much more rapidly than EDC under the same conditions.     

Purification, characterization, and synergistic action of endoglucanases from Fusarium lini

Five endoglucanases (1,4-beta-D-glucan-glucanohydrolase, EC 3.2.1.4) were isolated from Fusarium lini. Endo I and II were purified by preparative gel electrophoresis and Endo III, IV, and V were purified in a single-step procedure involving preparative flat-bed isoelectric focusing. All the endoglucanases were homogenous on disk gel electrophoresis and analytical isoelectric focusing in polyacrylamide gel. The pi values were between 6 and 6.6 for Endo III, IV, and V; for Endo I, the pi value was 8. The molecular weights of the enzymes were between 4 x 10(4) and 6.5 x 10(4). The K(m) values for endoglucanases using carboxymethyl cellulose (CM-cellulose) as the substrate were 2-12 mg/mL. The specificity of the enzymes was restricted to beta-1, 4-linkages. All the enzymes showed activity towards D-xylan. The endoglucanases had high viscosity reducing activity with CM-cellulose. Striking synergism was observed for the hydrolysis of CM-cellulose by endoglucanases. Endo II, IV, and V attacked cellopentaose and cellotetraose more readily than cellotriose. Endo II and V hydrolyzed cellotriose, cellotetraose, and cellopentaose, yielding a mixture of cellobiose with a trace amount of glucose; endo IV produced only cellobiose.     

Chemical modifications of the sigma subunit of the E. coli RNA polymerase.

The function of arginine, cysteine and carboxylic amino acid (glutamic and aspartic) residues of sigma was studied using chemical modification by group specific reagents. Following modification of 3 arginine residues with phenylglyoxal or 3 cysteine residues with N-ethylmaleimide (NEM) sigma activity was lost. Analysis of the kinetic data for inactivation indicated that one arginine or cysteine residue is essential for sigma activity. At low NEM concentration alkylation was limited to a non-critical cysteine which was identified as cysteine-132. Modification of arginine or cysteine residues had no observable effect on the binding of the inactivated sigma to the core polymerase. Modification of aspartic and/or glutamic acid residues with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) or 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMC) resulted in loss of sigma activity. The inactivation data indicated that one carboxylic amino acid residue is essential for sigma activity. Sigma modified with EDC, CMC or EDC in the presence of glycine was inactive in supporting promoter binding and initiation by core polymerase. Reaction with EDC plus (3H)glycine resulted in the incorporation of glycine into sigma. The (3H)glycine-sigma was unable to form a stable holoenzyme complex.     

色氨酸残基在内切葡聚糖酶分子中的作用

内切葡聚糖酶的化学修饰研究表明：色氨酸残基可能位于活性位点,与底物结合有关．荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对ｐＨ变化非常敏感,降低ｐＨ导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与ｐＨ诱导不同的构象变化．     

Modification of essential carboxyl group in rabbit muscle phosphorylase by water-soluble carbodiimide

Water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC) and glycine ethyl ester (GEE) as a nucleophile were used to modify the essential carboxyl group of phosphorylases. The inactive b form of the muscle phosphorylase was modified faster than the active a form and potato phosphorylases. Use of N,N,N',N'-tetramethyl-ethylenediamine (TEMED)-HCl buffer system (pH 6.2) resulted in a remarkable difference from the previous results obtained with phosphate and beta-glycerophosphate buffer systems. That is, the substrate glucose 1-phosphate gave the best protection of the three phosphorylase activities. Glucose and glycogen were also effective to retard the inactivation of muscle phosphorylases, though glycogen was not effective for the potato enzyme. The EDC-GEE-modified phosphorylase b retained the affinity for AMP-Sepharose, though partially modified enzyme completely lost the homotropic cooperativity. Phosphorylase b was subjected to differential labeling with [14C]GEE. A labeled peptide was obtained after CNBr cleavage and peptic digestions, and corresponded to the catalytic site sequence surrounding the GEE-substituted Asp 661 and Glu 664. Either or both of these EDC-modified carboxyl residues may have an important role in the catalytic reaction.     

Differential reactivity of active sites in human plasma cholinesterase toward 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Human plasma cholinesterase was found to be inhibited by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a biphasic manner. The faster phase of the inhibition led to loss of approximately 50% of the activity (measured at pH 7.0, 30 degrees C, using 2.5 mM butyrylthiocholine) and was irreversible. Inhibition in the slower phase was reversible by 0.25 M hydroxylamine. The protective effect of 1 mM propranolol indicated that the target residue in both phases was localized at the active site. Lineweaver-Burk plots for butyrylthiocholine were obtained at different times during the course of inactivation. It was found that for both native and partially inactivated enzymes the plots could be analyzed in terms of two activities showing hyperbolic saturation with the substrate, with Km values of 0.055 +/- 0.015 and 2.0 +/- 0.2 mM. The carbodiimide affected the maximal velocities of the component activities, leaving the Km's unchanged. The low-Km component was lost in the first phase of the inactivation. The loss of the high-Km component paralleled the second phase. It was concluded that the active sites in the tetrameric enzyme form two classes, differing in their affinity for butyrylthiocholine and their susceptibility to inhibition by the active site-directed carbodiimide.     

Kinetic study on chemical modification of taka-amylase A. I. Location and role of tryptophan residues

1. Five and four tryptophan residues in Taka-amylase A [EC 3.2.1.1] of A. oryzae (TAA) were modified with dimethyl(2-hydroxy-5-nitrobenzyl)-sulfonium bromide (K-IWS) in the absence and the presence of 15% maltose (substrate analog), respectively. Only one tryptophan residue was modified with dimethyl(2-methoxy-5-nitrobenzyl)-sulfonium bromide (K-IIWS) irrespective of the presence or absence of maltose. Kinetic parameters (molecular activity, k0, Michaelis constant, Km, and inhibitor constant, Ki) of the enzyme modified with K-IWS and K-IIWS were determined. The k0 value decreased with increase in the number of modified residues, but Km and Ki values and the type of inhibition were not altered by the modification. 2. The fluorescence quenching reaction of TAA with N-bromosuccinimide (NBS) proceeded in three phases. The second-order rate constants of the three phases were determined to be (4.3 +/- 0.5) x 10(5) M-1 . s-1, (2.1 +/- 0.3) x 10(3) M-1 . s-1 and (1.7 +/- 0.2) x 10(2) M-1 . s-1, respectively. In the presence of maltose, the first phase was further separated into two phases with rate constants of (4.6 +/- 0.6) x 10(6) M-1 . s-1 and (6.9 +/- 1.1) x 10(4) M-1 . s-1, respectively. On the basis of the results, it is estimated that five out of nine tryptophan residues are accessible to the solvent and among them, two tryptophan residues are substantially exposed: one is located in the maltose binding site near the catalytic site (its modification affects the catalytic function), and the other exists on the enzyme surface far from the active site.     

Inactivation of human fibroblast collagenase by chloroacetyl N-hydroxypeptide derivatives.

When human fibroblast collagenase was incubated with ClCH2CO-(N-OH)Leu-Ala-Gly-NH2 (2-5 mM) in Tris buffer, pH 7.4 at 25 degrees C, a slow, time-dependent inhibition of the enzyme was observed. Dialysis against a buffer to remove free inhibitor did not reactivate the enzyme. A reversible competitive inhibitor, phthaloyl-GlyP-Ile-Trp-NHBzl (50 microM) partially protected the enzyme from inactivation by the compound. From the concentration dependent rates of inactivation Ki = 0.5 +/- 0.1 mM and k3, the rate constant for inactivation = 3.4 +/- 0.3 x 10(-3) min-1 were determined. The inactivation followed the pH optimum (6.5-7.0) for the enzyme activity, suggesting direct involvement of the same active site residue(s). The reaction mode of the inhibitor may be analogous to that of the inactivation of Pseudomonas aeruginosa elastase [Nishino, N. and Powers, J. (1980) J. Biol. Chem., 255, 3482] in which the catalytic glutamate carboxyl was alkylated by the inhibitor after its binding to enzyme through the hydroxamic Zn2+ ligand. All carboxyl groups in the inactivated collagenase were modified with 0.1 M ethyl dimethylaminopropyl carbodiimide/0.5 M glycinamide in 4 M guanidine at pH 5. The inactivator-affected carboxyl group was then regenerated with 1 M imidazole at pH 8.9, 37 degrees C for 12 h and the protein was radiolabeled with 3H-glycine methyl ester and carbodiimide to incorporate 0.9 residue glycine per mol enzyme.     

Evidence for a critical glutamyl and an aspartyl residue in the function of pig heart diphosphopyridine nucleotide dependent isocitrate dehydrogenase.

The pH dependence of the maximum velocity of the reaction catalyzed by diphosphopyridine nucleotide (DPN) dependent isocitrate dehydrogenase indicates the requirement for the basic form of an ionizable group in the enzyme-substrate complex with a pK of 6.6. This pK is unaltered from 10 to 33 degrees C, suggesting the ionization of a carboxyl rather than an imidazolium ion. The enzyme is inactivated upon incubation with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide in the presence of glycinamide or glycine ethyl ester. This inactivation is dependent on pH and the rate constant (k) increases as the pH is decreased in the range 7.3 to 6.25. A plot of 1/(H+) vs. 1/k suggests that the enzyme is inactivated as a result of the modification of a single ionizable group in this pH range. The coenzyme DPN and substrate alpha-ketoglutarate do not affect the rate of inactivation. In contrast, manganous ion (2 mM) and isocitrate (60 mM) produce a sevenfold decrease in the rate constant. The allosteric activator ADP (1 mM) does not itself influence the rate of inactivation; however, it reduces the concentration of Mn2+ (1 mM) and isocitrate (20 mM) required to produce the same decrease in the inactivation constant. These observations imply that the modification occurs at the substrate-binding site. Experiments employing [1-14C]glycine ethyl ester show a net incorporation of 2 mol of glycine ethyl ester per subunit (40 000), concomitant with the complete inactivation of the enzyme. The radioactive modified enzyme, after removal of excess reagent by dialysis, was exhaustively digested with proteolytic enzymes. High voltage electrophoretic analyses of the hydrolysate at pH 6.4 and 3.5 yield two major radioactive spots with approximately equal intensity, which correspond to gamma-glutamylglycine and beta-aspartylglycine, the ultimate products of reaction with glutamic and aspartic acids, respectively. Modification in the presence of manganous ion and isocitrate results in significant reduction in the incorporation of radioactivity into the two dipeptides. These results suggest that carbodiimide attacks one glutamyl and one aspartyl residue per subunit of the enzyme and that the integrity of these residues is crucial for the enzymatic activity.     

Carboxyl groups at the two active centers of sucrase-isomaltase from rabbit small intestine.

1. Seveal selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation only in the reaction of carboxyl groups with carbodiimide (+lycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem 251,3250--3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed.     

Evidence for tryptophan in proximity to histidine and cysteine as essential to the active site of an alkaline protease

The presence, microenvironment, and proximity of an essential Trp with the essential His and Cys residues in the active site of an alkaline protease have been demonstrated for the first time using chemical modification, chemo-affinity labeling, and fluorescence spectroscopy. Kinetic analysis of the N-bromosuccinimide- (NBS) or p-hydroxymercuribenzoate- (PHMB) modified enzyme from Conidiobolus sp. revealed that a single Trp and Cys are essential for activity in addition to the Asp, His, and Ser residues of the catalytic triad. Full protection by casein against inactivation of the enzyme by NBS and quenching of Trp fluorescence upon binding of the enzyme with NBS, substrate (sAAPF-pNA), or inhibitor (SSI) confirmed participation of the Trp residue at the substrate/inhibitor binding site of the alkaline protease. Comparison of the K(sv) values for the charged quenchers CsCI (1.66) and KI (7.0) suggested that the overall Trp microenvironment in the protease is electropositive. The proximity of Trp with His was demonstrated by the sigmoidal shape of the pH-dependent fluorometric titration curve with a pK(F) of 6.1. The vicinity of Trp with Cys was indicated by resonance energy transfer between the intrinsic fluorophore (Trp) and 5-iodoacetamide-fluorescein labeled Cys (extrinsic fluorophore). Our results on the proximity of Trp with essential His and Cys thus confirm the presence of Trp in the active site of the alkaline protease.     

Isolation and characterization of an endo-(1,4)-{beta}-glucanase secreted by Achlya ambisexualis

Models of wall loosening in fungi and other walled eukaryotes require the action of proteins able to reduce the degree of linkage between components of the wall. In the oomycete Achlya ambisexualis, such a role has been proposed for a suite of endoglucanases that are secreted during branching and during the measurable wall softening associated with osmotic stress. We report here the isolation and characterization of one of these isoenzymes. The enzyme has a molecular weight of 32 kDa, a pH optimum of 6.75, a pI of 4.5, and a temperature optimum of 35 C. It is partially inhibited by sulfhydryl-binding reagents and completely inhibited by the tryptophan-binding reagent NBS. The enzyme has an endohydrolytic mode of action with substrate specificity towards glucans that contain β-(1,4) linkages, either alone (carboxymethyl cellulose) or as mixed linkage (1,4-1,3)-β-glucans (e.g., Avena glucan). It does not, however, degrade amorphous insoluble (phosphoric acid swollen) cellulose. Most significantly, the enzyme can also hydrolyze linkages in an Achlya cell wall fraction previously shown to consist of a mixed-linkage (1,4-1,3)-β-glucan. This property is consistent with the long-standing hypothesis that the branching-related endoglucanases of oomycetes play a role in cell wall loosening.     

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed.     

Effect of prior treatment with Clostridium perfringens epsilon toxin inactivated by various agents on lethal, pressor and contractile activities of the toxin

Lethal and pressor activities, and the contractile responses of rat isolated ileum to Clostridium perfringens epsilon toxin, were significantly prevented by the prior administration of epsilon toxin inactivated by 1-ethyl-3-(3-diethyl-aminopropyl) carbodiimide in the presence of glycine methyl ester (EDC), 2,4,6-trinitrobenzene sulfonic acid (TNBS), succinic anhydride (SA) and ethoxyformic anhydride (EFA). However, the prior administration of the toxin inactivated by N-acetylimidazole (NAI), tetranitromethane (TNM) and N-bromosuccinimide (NBS) resulted in no inhibition of these biological activities. These data suggest that the toxin interacts with specific site(s) on target organs or tissues. The relationship between amino acid residues and the actions of the toxin is described.     

Chemical modification of xylanase from Trichosporon cutaneum shows the presence of carboxyl groups and cysteine residues essential for enzyme activity

The endo--1,4-xylanase (EC 3.2.1.8) from Trichosporon cutaneum was chemically modified using amino acid-specific reagents. The enzyme does not bear arginines essential for activity, since 1,2-cyclohexanedione and 2,3-butanedione, although they modify the enzyme (after chromatographic analysis), have no effect on its activity. Reaction of the enzyme with tetranitromethane and N-acetylimidazole did not result in a significant activity loss as a result of modification of tyrosine residues. The water-soluble carbodiimide 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide inactivated the xylanase rapidly and completely in a pseudo-first-order process, and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. A mixture of neutral xylooligomers provided significant protection of the enzyme against this carbodiimide inactivation. Reaction of the xylanase with 2,4,6-trinitrobenzene sulfonic acid did not result in a significant activity loss as a result of modification of lysine residues. Titration of the enzyme with 5,5-dithiobis-(2-nitrobenzoic acid) and treatment with iodoacetamide and p-chloromercuribenzoate indicated the presence of a free/active thiol group. Xylan completely protected the enzyme from inactivation by p-hydroxymercuribenzoate, suggesting the presence of cysteine at the substrate-binding site. Inactivation of xylanase by p-hydroxymercuribenzoate could be restored by cysteine.