Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum

Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum plays a central role in the newly discovered acetyl-CoA pathway [Wood, H.G., Ragsdale, S.W., & Pezacka, E. (1986) FEMS Microbiol. Rev. 39, 345-362]. The enzyme catalyzes the formation of acetyl-CoA from methyl, carbonyl, and CoA groups, and it has specific binding sites for these moieties. In this study, we have determined the role of tryptophans at these subsites. N-Bromosuccinimide (NBS) oxidation of the exposed and reactive tryptophans (5 out of a total of approximately 20) of CODH at pH 5.5 results in the partial inactivation of the exchange reaction (approximately 50%) involving carbon monoxide and the carbonyl group of the acetyl-CoA. Also, about 70% of the acetyl-CoA synthesis was abolished as a result of NBS modification. The presence of CoA (10 microM) produced complete protection against the partial inhibition of the exchange activity and the overall synthesis of acetyl-CoA caused by NBS. Additionally, none of the exposed tryptophans of CODH was modified in the presence of CoA. Ligands such as the methyl or the carbonyl groups did not afford protection against these inactivations or the modification of the exposed tryptophans. A significant fraction of the accessible fluorescence of CODH was shielded in the presence of CoA against acrylamide quenching. On the basis of these observations, it appears that certain tryptophans are involved at or near the CoA binding site of CODH.     

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.     

Isoamylamine as the Possible Neuroactive Metabolite of l-Leucine

The states of tryptophan residues in Abrus precatorius agglutinin (APA) were analyzed by chemical modification and solvent perturbation UV-difference spectroscopy. The number of tryptophan residues available for N-bromosuccinimide (NBS) oxidation increased with lowering pH, and 20 out of the 24 tryptophans in APA were modified at pH 3.0, while 2 tryptophans were eventually oxidized at pH 5.0. Modification of tryptophan greatly decreased the binding of APA with saccharides, and only 4% of the hemagglutinating activity was retained after modification of 4 tryptophan residues/molecule. When the modification was done in the presence of lactose or galactose, 2 tryptophan residues/molecule remained unmodified with a retention of a fairly high hemagglutinating activity. The data from solvent perturbation UV-difference spectroscopy indicated that 6 tryptophans were on the surface of the APA molecule, and 4 tryptophan residues/molecule were shielded from the perturbing effect of the solvent upon binding with lactose.

Based on these results, we proposed that in the saccharide-binding site on each B-chain of APA there exists one tryptophan residue directly involved in saccharide binding, and near the binding site there is another tryptophan residue whose state is also changeable upon binding with saccharide.     

Binding and reversibility of Thermobifida fusca Cel5A,Cel6B,and Cel48A and their respective catalytic domains to bacterial microcrystalline cellulose

The binding and reversibility of Thermobifida fusca intact Cel5A, Cel5B, and Cel48A and their corresponding catalytic domains (CDs) to bacterial microcrystalline cellulose (BMCC) were studied at 5 degrees C. The binding of the intact cellulases and of corresponding CDs to BMCC was irreversible in all regions: Langmuir binding (region I), interstice penetration (region II), and interstice saturation (region III). The three cellulose binding domains (CBMs) bind reversibly in "region I" although their respective CDs do not. The irreversible binding of these enzymes in the Langmuir region does not satisfy the Langmuir assumption; however, the overall fit of the Interstice Saturation model, which includes binding in MBCC interstices as well as on the freely accessible surface (Jung et al., 2002a) is good. The main limitation of the model is that it does not explicitly address a mechanism for forming the enzyme-substrate complex within the active site of the CDs.     

Microenvironment of tryptophan residues in β-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes

The changes of microenvironment of tryptophan residues in β-lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) β-lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM β-lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.     

Pea lectin unfolding reveals a unique molten globule fragment chain

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains -a long β-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a β-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ∼ 20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule β-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.     

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.     

Terbium(III) luminescence study of tyrosine emission from Escherichia coli glutamine synthetase.

Radiationless energy transfer from tyrosine to Tb(III) in Escherichia coli glutamine synthetase and its two mutants (W57L and W158S) has been utilized to assess the tyrosine residue(s) responsible for the observed tyrosine emission and to investigate its spatial relationships to the two metal binding sites of GS. The interference from tryptophan fluorescence was removed by chemical modification of the tryptophan residues by N-bromosuccinimide (NBS). The Tyr-Tb(III) distances measured by using F?rster energy-transfer theory were in good agreement among the three enzymes with average distances of 10.7 and 11.2 A from Tyr to the two metal binding sites. The pKa value for the ionization of tyrosine was determined from fluorescence titration experiments to be approximately 10 for both mutant enzymes. The similarities in pKa values and Tyr-Tb(III) distances observed for all three enzymes lead to the conclusion that the same tyrosine residue(s), is (are) most likely responsible for the Tyr emission. According to the crystal structure distances from tyrosine residues to the two metal binding sites of GS, it is believed that Tyr-179 is the main contributor to the observed Tyr emission. The fact that an intense Tyr emission was observed for W57L GS but not for W158S GS indicates that Trp-57 is much more effective than Trp-158 in quenching the Tyr-179 emission probably through a F?rster-type energy transfer. Furthermore, modification of Trp-57 by NBS causes no significant increase in Tyr-179 emission while replacement of Trp-57 by leucine does. This may indicate that oxidized Trp-57 is also an effective quencher for Tyr-179 emission.     

Microenvironment of tryptophan residues in beta-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes.

The changes of microenvironment of tryptophan residues in -lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) -lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM -lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.     

Chemical Modification of Tryptophan Residues in Castor Bean Hemagglutinin

Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that the extent of modification of tryptophan in CBH is influenced by an ionizable group with pK_a = 3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-chain of CBH.     

Modification of a single tryptophan of the inorganic pyrophosphatase from thermophilic bacterium PS-3: possible involvement in its substrate binding.

The effect of N-bromosuccinimide (NBS) on the activity of the inorganic pyrophosphatase (PPiase) from thermophilic bacterium PS-3 was studied. The enzyme was almost completely inactivated on chemical modification with NBS, depending upon the concentration of NBS. The presence of a complex of Mg2+ and a substrate analogue, imidodiphosphate (PNP), provided extensive protection against the inactivation, whereas Mg2+ or PNP alone showed no protective effect. Amino acid analysis of the NBS-modified enzyme after hydrolysis with 6 M HCl indicated no change in the amino acid composition. However, the magnetic circular dichroism (MCD) bands around 293 nm due to the tryptophan residue and the optical density at 280 nm, decreased concomitantly with modification by NBS. These results strongly suggested that the tryptophan residue at position 143, which is the only tryptophan residue per subunit in the thermophilic PPiase (Ichiba, T., Takenaka, O., Samejima, T. and Hachimori, A. (1990) J. Biochem. 108, 572-578), might be involved in the active site or be located in the vicinity of the active site. The circular dichroism (CD) spectrum in the far ultraviolet region showed no significant alteration during the modification, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modification considerably altered the CD bands in, the near ultraviolet region, indicating that a conformational change occurred in the vicinity of the active site in the enzyme molecule.     

Two forms of DNA polymerase delta from mouse cells. Purification and properties

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).     

Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.

The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.     

Identification of the Tryptophan Residue Located at the Substrate-binding Site of Rye Seed Chitinase-c

Chemical modifications of rye seed chitinase-c (RSC-c) with various reagents suggested the involvements of tryptophan and glutamic/aspartic acid residues in the activity. Of these, the modification of tryptophan residues with N-bromosuccinimide (NBS) was investigated in detail.

In the NBS-oxidation at pH 4.0, two of the six tryptophan residues in RSC-c were rapidly oxidized and the chitinase activity was almost completely lost. On the other hand, in the NBS-oxidation at pH 5.9, only one tryptophan residue was oxidized and the activity was greatly reduced. Analyses of the oxidized tryptophan-containing peptides from the tryptic and chymotryptic digests of the modified RSC-c showed that two tryptophan residues oxidized at pH 4.0 are Trp72 and Trp82, and that oxidized at pH 5.9 is Trp72.

The NBS-oxidation of Trp72 at pH 5.9 was protected by a tetramer of N-acetylglucosamine (NAG₄), a very slowly reactive substrate for RSC-c, and the activity was almost fully retained. In the presence of NAG4, RSC-c exhibited an UV -difference spectrum with maxima at 284 nm and 293 nm, attributed to the red shift of the tryptophan residue, as well as a small trough around 300 nm probably due to an alteration of the environment of the tryptophan residue. From these results, it was suggested that Trp72 is exposed on the surface of the RSC-c molecule and involved in the binding to substrate.