Substrate binding subsites of chitinase from barley seeds and lysozyme from goose egg white.

Substrate binding subsites of barley chitinase and goose egg white lysozyme were comparatively investigated by kinetic analysis using N-acetylglucosamine oligosaccharide as the substrate. The enzymatic hydrolysis of hexasaccharide was monitored by HPLC, and the reaction time-course was analyzed by the mathematical model, in which six binding subsites (B, C, D, E, F, and G) and bond cleavage between sites D and E are postulated. In this model, all of the possible binding modes of substrate and products are taken into consideration assuming a rapid equilibrium in the oligosaccharide binding processes. To estimate the binding free energy changes of the subsites, time-course calculation was repeated with changing the free energy values of individual subsites, until the calculated time-course was sufficiently fitted to the experimental one. The binding free energy changes of the six binding subsites, B, C, D, E, F and G, which could give a calculated time-course best fitted to the experimental, were 0.0, -5.0, +4.1, -0.5, -3.8, and -2.0 kcal/mol for barley chitinase, and -0.5, -2.2, +4.2, -1.5, -2.6, and -2.8 kcal/mol for goose egg white lysozyme. The binding mode predicted from the p-nitrophenyl-penta-N-acetylchitopentaoside splitting pattern for each enzyme was also analyzed by the identical subsite model. Using the free energy values listed above, the binding mode distribution calculated was fitted to the experimental with a slight modification of free energy value at site G. We concluded that the binding subsite model described above reflects the substantial mechanism of substrate binding for both enzymes. The relatively large disparity in free energy value at site C between these enzymes may be due to the different secondary structures of polypeptide segments interacting with the sugar residue at site C.

Specificity of chitosanase from Bacillus pumilus.

Partially (25-35%) N-acetylated chitosan was digested by chitosanase from Bacillus pumilus BN-262, and structures of the products, partially N-acetylated chitooligosaccharides, were analyzed in order to investigate the specificity of the chitosanase. The chitosanase produced glucosamine (GlcN) oligosaccharides abundantly, indicating that the chitosanase splits the beta-1,4-glycosidic linkage of GlcN-GlcN. The chitosanase also produced hetero-oligosaccharides consisting of glucosamine and N-acetyl-D-glucosamine (GlcNAc). Three types of the hetero-oligosaccharides purified by cation-exchange chromatography and HPLC were found to have GlcNAc residue at their reducing end and GlcN residue at their non-reducing end, indicating that the chitosanase can also split the linkage of GlcNAc-GlcN. The determination of the mode of action toward partially N-acetylated chitosan enables a classification of chitosanases according to their specificities and a more precise definition of chitosanases.

Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization.

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.

Chitinolytic enzymes: catalysis, substrate binding, and their application.

After the epoch-making report on X-ray crystal structure of a lysozyme-N-acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 +) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate-assisted mechanism. Hen egg white lysozyme has the binding cleft represented by (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)(-1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application.

Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments.

Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of D-glucosamine oligosaccharides ((GlcN)n, n = 3, 4, 5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (Tm) of E22Q was found to be 44.4 degrees C at pH 7.0. However, the Tm increased upon the addition of (GlcN), by 1.3 degrees C (n = 3), 2.5 degrees C (n = 4), 5.2 degrees C (n = 5), or 7.6 degrees C (n = 6). No appreciable change in Tm was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect Tm of the protein. Thus, we concluded that the stabilization effect of (GlcN)n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in Tm induced by (GlcN)6 binding were 2.7 degrees C for E22D and 4.2 degrees C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.

Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides.

The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).

Properties of Manduca sexta chitinase and its C-terminal deletions.

Manduca sexta (tobacco hornworm) chitinase is a molting enzyme that contains several domains including a catalytic domain, a serine/threonine-rich region, and a C-terminal cysteine-rich domain. Previously we showed that this chitinase acts as a biopesticide in transgenic plants where it disrupts gut physiology. To delineate the role of these domains further and to identify and characterize some of the multiple forms produced in molting fluid and in transgenic plants, three different forms with variable lengths of C-terminal deletions were generated. Appropriately truncated forms of the M. sexta chitinase cDNA were generated, introduced into a baculovirus vector, and expressed in insect cells. Two of the truncated chitinases (Chi 1-407 and Chi 1-477) were secreted into the medium, whereas the one with the longest deletion (Chi 1-376) was retained inside the insect cells. The two larger truncated chitinases and the full-length enzyme (Chi 1-535) were purified and their properties were compared. Differences in carbohydrate compositions, pH-activity profiles, and kinetic constants were observed among the different forms of chitinases. All three of these chitinases had some affinity for chitin, and they also exhibited differences in their ability to hydrolyze colloidal chitin. The results support the hypothesis that multiple forms of this enzyme occur in vivo due to proteolytic processing at the C-terminal end and differential glycosylation.

Separation and mutarotation of anomers of chitooligosaccharides.

In the course of a study on the lysozyme-catalyzed reaction of chitooligosaccharides, it was found that each chitooligosaccharide gave two completely separated peaks on high-performance liquid chromatography with a partition column. Synthetic 2-acetamido-2-deoxy-beta-D-glucopyranose gave [alpha] D14 = -18.1 degrees (c = 0.51, H2O) and a large second peak with a minor first peak on high-performance liquid chromatography. When an aqueous solution of the beta-anomer was allowed to stand, the area of the first peak on high-performance liquid chromatography increased, together with a decrease in the area of the second peak and an increase in [alpha] D value. It was concluded that the two peaks of each chitooligosaccharide on high-performance liquid chromatography were due to the separation of alpha- and beta-anomers. The mutarotation of 2-acetamido-2-deoxy-beta-D-glucopyranose was followed by monitoring the [alpha] D value and in the peak area of the two peaks on high-performance liquid chromatography. It was found that the ratios of alpha- and beta-anomers of chitooligosaccharides produced by the lysozyme-catalyzed reaction of chitopentose were different from those of the corresponding authentic chitooligosaccharides which were allowed to stand in the absence of the enzyme under the conditions used for the enzymatic reaction.

Lysozyme-catalyzed reaction in continuous flow system.

The lysozyme-catalyzed reaction of chitooligosaccharide was carried out in a continuous flow system in which the solution of substrate, chitooligosaccharide [(GlcNAc)n], flowed into the lysozyme solution in an ultrafiltration apparatus and the products were filtered off. The filtrate was continuously collected in test tubes with the aid of a fraction collector. The product distribution in each fraction was analyzed by high performance gel filtration. Using (GlcNAc)5 as the substrate, the concentrations of products, (GlcNAc)1----4, increased gradually and came to the steady state when the volume of the outflow amounted to sixfold of the inside volume. Before reaching the steady state, the product distribution was quite different from that observed in the closed reaction system, in which the reaction species are not exchangeable through the boundary of the system. The outflows of (GlcNAc)3-5 were delayed in comparison with those of GlcNAc and (GlcNAc)2. The delay period increased with the decrease in substrate concentration, and was shortened by using the [Asp 101 or Trp 62]-modified lysozyme instead of the native lysozyme. These results suggest that the delay in the (GlcNAc)3-5 outflows is caused by the nonproductive binding of the oligosaccharide to the lysozyme molecule. The profile of the flow reaction yields information not only on the catalytic efficiency but also on the substrate binding efficiency of the lysozyme.

Enzymatic activity of Trp 62-modified lysozyme.

The time-courses of action of Trp 62-modified lysozymes on the initial substrate chitopentaose were measured by means of high-performance gel-filtration. The activities of the modified lysozymes, represented by the rate of disappearance of the initial substrate (overall rate) were lowered to various extents depending on the method of the modification. On the other hand, the time-courses were calculated by changing the values of rate constants, using the binding free energy of each subsite estimated by the optimization technique (Kuhara et al. (1982) J. Biochem.). For NBS- and NPS-lysozymes, the calculated time-courses were not in good agreement with the experimental ones, when the binding free energies estimated by the optimization technique were used for the calculation. Therefore, the binding free energies of the subsites were estimated from the experimental time-courses with the assumption that the values of the rate constants do not change upon modification of Trp 62 in subsite C. As a result, it was found that, though the modification was at subsite C, the binding free energy of subsite A was profoundly lowered, while that of subsite B remained almost unchanged.

Estimation of the free energy change of substrate binding lysozyme-catalyzed reactions.

The binding constant and binding free energy of each subsite of lysozyme upon substrate binding have been customarily estimated from the experimental data with assumptions regarding the binding mode of substrate and the additivity of binding free energy of each subsite. In the present study, the binding constants and binding free energy of subsites were estimated from experimentally obtained overall binding constants on native and Trp 62-modified lysozymes. The estimations of binding constants and binding free energy were carried out by an optimization method, the modified Powell method, without assuming the binding mode for substrate. First the binding free energies of subsites A, B, and C were estimated from the experimental binding constants of (GlcNAc)1 to (GlcNAc)3, and the binding free energies of subsites D, E, and F were determined from the estimated free energies of subsites A, B, and C, and the experimentally obtained reaction time-courses of substrate (GlcNAc)5. Finally, the values of three rate constants in the lysozyme-catalyzed reaction of chitooligosaccharide were estimated from the experimental time-course by using the binding free energies obtained by the modified Powell method.

Human lysozyme-catalyzed reaction of chitooligosaccharides.

The time-courses of the human lysozyme-catalyzed reaction of chitopentaose were measured by high-performance gel-filtration in comparison with those of hen egg-white lysozyme. Human lysozyme has considerably larger rate constants for the cleavage of glycosidic linkages and transglycosylation than those of hen lysozyme, in agreement with the fact that human lysozyme exhibits a large lytic activity. It has been reported that binding subsite D in human lysozyme has negative free energy on substrate binding, whereas subsite D in hen lysozyme has unfavorable positive free energy due to the distortion of a sugar residue. The time-courses calculated under the assumption that subsite D in human lysozyme has negative free energy on substrate binding did not fit the experimentally obtained time-courses, even though the combination of values of rate constants in the enzymatic reaction widely varied in the calculation of the time-courses. Thus, it was concluded that subsite D in human lysozyme may not have negative binding free energy, but positive values similar to hen lysozyme.

State of binding subsites in Asp 101-modified lysozymes.

The environments of the binding subsites in Asp 101-modified lysozyme, in which glucosamine or ethanolamine is covalently bound to the carboxyl group of Asp 101, were investigated by chemical modification and nuclear magnetic resonance spectroscopy. Trp 62 in each of the native and the modified lysozymes was nitrophenylsulfenylated. The yield of the nitrophenylsulfenylated derivative from the lysozyme modified with glucosamine at Asp 101 (GlcN-lysozyme) was considerably lower than those from native lysozyme and from the lysozyme modified with ethanolamine at Asp 101 (EtN-lysozyme). These results suggest that Trp 62 in GlcN-lysozyme is less susceptible to nitrophenylsulfenylation. Kinetic analyses of the [Trp 62 and Asp 101]-doubly modified lysozymes indicated that the nitrophenylsulfenylation of Trp 62 in the native lysozyme, EtN-lysozyme, or GlcN-lysozyme decreased the sugar residue affinity at subsite C while increasing the binding free energy change by 2.7 kcal/mol, 1.5 kcal/mol, or 0.1 kcal/mol, respectively. Although the profile of tryptophan indole NH resonances in the 1H-NMR spectrum for EtN-lysozyme was not different from that for the native lysozyme, the indole NH resonance of Trp 62 in GlcN-lysozyme was apparently perturbed in comparison with that of native lysozyme. These results suggest that the environment of subsite C in GlcN-lysozyme is considerably different from those in native lysozyme and EtN-lysozyme. The glucosamine residue attached to Asp 101 may contact the sugar residue binding site of the lysozyme, affecting the environment of subsite C.

Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.

Enzymatic activity of avian egg-white lysozymes.

The experimental time-courses of eight avian lysozymes, seven hen-type lysozymes and one goose-type lysozyme, were measured with a substrate of chitopentaose (GlcNAc)5 at pH 5.0 and 50 degrees C. Chitooligosaccharides in the reaction mixture were analyzed by high-performance gel-filtration. From the experimental time-courses, the overall reaction rates represented by the disappearance of the initial substrate and the values of reaction parameters were estimated by computer analysis. With taking hen lysozyme as the reference, the values of reaction parameters estimated were correlated to the replaced amino acid residue in the binding site of the lysozyme, and the roles of some amino acid residues in the binding site were discussed.

Exchange of nucleoside monophosphate-binding domains in adenylate kinase and UMP/CMP kinase.

Two types of active chimeric enzymes have been constructed by genetic engineering of chicken cytosolic adenylate kinase (AK) and porcine brain UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-binding domain of AK in the remaining body of UCK; and the other, designated as AUA, carries a UMP/CMP-binding domain of UCK in the remaining body of AK. Steady-state kinetic analysis of these chimeric enzymes revealed that UAU is 4-fold more active for AMP, 40-fold less active for UMP, and 4-fold less active for CMP than the parental UCK, although AUA has considerably lowered reactivity for both AMP and UMP. Circular dichroism spectra of the two chimeric enzymes suggest that UAU and AUA have similar folding structures to UCK and AK, respectively. Furthermore, proton NMR measurements of the UCK and UAU proteins indicate that significant differences in proton signals are limited to the aromatic region, where an imidazole C2H signal assigned to His31 shows a downfield shift upon conversion of UCK to UAU, and the signals assigned to Tyr49 and Tyr56 in the UMP/CMP-binding domain disappear in UAU. In contrast, AUA has a Tm value about 11 degreesC lower than AK, whereas UAU and UCK have similar Tm values. These results together show that the substrate specificity of nucleoside monophosphate (NMP) kinases can be engineered by the domain exchange, even though the base moiety of NMP appears to be recognized cooperatively by both the NMP-binding domain and the MgATP-binding core domain.

Lysozyme-catalyzed reaction of chitooligosaccharides.

The time-courses of substrate consumption and product formation in the lysozyme-catalyzed reaction were determined with (GlcNAc)4 and (GlcNAc)5 as substrate to accumulate data suitable for the estimation of rate constants by numerical analysis. The lysozyme-catalyzed reactions were followed by TLC or HPLC. (GlcNAc)4 decomposed apparently to small oligosaccharides within 5 h, and (GlcNAc)5 decomposed within 15 min at pH 5.0 and 50 degrees C. The temperature-dependence of the rate of disappearance of the initial substrate showed a different profile from that observed with glycol chitin as substrate by the reducing power method. The order (or distribution) of the amount of product formed from (GlcNAc)5 in the reaction time-course determined by TLC differed from that determined by HPLC. The relative error in HPLC was much less than that in TLC, and the time-course determined by HPLC was thought to be of sufficient accuracy for the estimation of rate constants by computer analysis.

Estimation of rate constants in lysozyme-catalyzed reaction of chitooligosaccharides.

The rate constants of the cleavage of glycoside linkage, hydration (hydrolysis) and transglycosylation in a lysozyme-catalyzed reaction of substrate chitooligosaccharides were evaluated by computer analysis of the experimentally obtained reaction time-courses. In the computer analysis, the rate equation was numerically solved by use of the known binding constants for each subsite. Because of the complexity of the lysozyme-catalyzed reaction, optimal values of rate constants were determined by checking the sensitivity of each rate constant to the computed time-courses. It was not possible to estimate uniquely the rate constants for transglycosylation and hydration, owing to the nature of the enzymatic reaction, but it was possible to estimate accurately their ratio. The estimated values were 0.94 s-1 for the rate constant for the cleavage of glycosidic linkage and 133 for the ratio of rate constants of transglycosylation and hydration.

1H-NMR study on the structure of lysozyme from guinea hen egg white.

The structure of lysozyme from guinea hen egg white (GEWL), which differs from hen egg white lysozyme (HEWL) by ten amino acid substitutions, was investigated by nuclear magnetic resonance (NMR) spectroscopy. GEWL and HEWL were very similar to each other in their tertiary structure as judged from the profile of 1H-NMR spectra, pH titration, and an N-acetylglucosamine trisaccharide [(GlcNAc)3 binding experiment. However, we have noticed several characteristics which distinguish GEWL from HEWL. The signal of Trp 108 indole N1H of GEWL was shifted upfield by about 0.3 ppm when compared with that of HEWL, and its hydrogen exchange was faster than that of HEWL. The pKa values of Glu 35 estimated from the pH titration curve of Trp 108 indole N1H were different between GEWL and HEWL. From a careful examination of spectral changes caused by (GlcNAc)3 binding, the changes in the chemical shift values of Trp 28 C5H and Asn 59 alpha CH of GEWL were found to be slightly larger than those of HEWL. Ile 55 of HEWL is replaced by valine in GEWL. Such a replacement may affect the neighboring hydrogen bonding between the main chain C = O of Leu 56 and Trp 108 indole N1H, resulting in a change in the microenvironment of the substrate-binding site near Trp 108.

Chitosanase-catalyzed hydrolysis of 4-methylumbelliferyl beta-chitotrioside.

4-Methylumbelliferyl beta-chitotrioside [(GlcN)(3)-UMB] was prepared from 4-methylumbelliferyl tri-N-acetyl-beta-chitotrioside [(GlcNAc)(3)-UMB] using chitin deacetylase from Colletotrichum lindemuthianum, and hydrolyzed by chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)(3)-UMB was confirmed by (1)H-NMR spectroscopy and mass spectrometry. When the (GlcN)(3)-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)(3) itself cannot be hydrolyzed by the chitosanase, (GlcN)(3)-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The k(cat) and K(m) values obtained for the substrate (GlcN)(3)-UMB were determined to be 8.1 x 10(-5) s(-1) and 201 microM, respectively. From TLC analysis of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.