Modification Of Tryptophan Residues Research Articles

The C-Mannosylome of Human Induced Pluripotent Stem Cells Implies a Role for ADAMTS16 C-Mannosylation in Eye Development

C-mannosylation is a modification of tryptophan residues with a single mannose and can affect protein folding, secretion, and/or function. To date, only a few proteins have been demonstrated to be C-mannosylated, and studies that globally assess protein C-mannosylation are scarce. To interrogate the C-mannosylome of human induced pluripotent stem cells, we compared the secretomes of CRISPR–Cas9 mutants lacking either the C-mannosyltransferase DPY19L1 or DPY19L3 to WT human induced pluripotent stem cells using MS-based quantitative proteomics. The secretion of numerous proteins was reduced in these mutants, including that of A Disintegrin And Metalloproteinase with ThromboSpondin Motifs 16 (ADAMTS16), an extracellular protease that was previously reported to be essential for optic fissure fusion in zebrafish eye development. To test the functional relevance of this observation, we targeted dpy19l1 or dpy19l3 in embryos of the Japanese rice fish medaka (Oryzias latipes) by CRISPR–Cas9. We observed that targeting of dpy19l3 partially caused defects in optic fissure fusion, called coloboma. We further showed in a cellular model that DPY19L1 and DPY19L3 mediate C-mannosylation of a recombinantly expressed thrombospondin type 1 repeat of ADAMTS16 and thereby support its secretion. Taken together, our findings imply that DPY19L3-mediated C-mannosylation is involved in eye development by assisting secretion of the extracellular protease ADAMTS16.

Interaction of Cholesterol With the Human SLC1A5 (ASCT2): Insights Into Structure/Function Relationships.

The human SLC1A5 commonly known as ASCT2 is a sodium-dependent neutral amino acid antiporter involved in transmembrane traffic of glutamine that is exchanged through the cell membrane with smaller amino acids such as serine or threonine. Due to the strong overexpression in human cancers, ASCT2 is widely studied for its relevance to human health. Of special interest are the aspects related to the regulation of its function. The role of cholesterol as a modulator of the transport activity has been studied using a combined strategy of computational and experimental approaches. The effect of cholesterol on the -[3H]glutamineex/glutaminein antiport in proteoliposomes has been evaluated by adding cholesteryl hemisuccinate. A strong stimulation of transport activity was observed in the presence of 75 μg cholesteryl hemisuccinate per mg total lipids. The presence of cholesterol did not influence the proteoliposome volume, in a wide range of tested concentration, excluding that the stimulation could be due to effects on the vesicles. cholesteryl hemisuccinate, indeed, improved the incorporation of the protein into the phospholipid bilayer to some extent and increased about three times the Vmax of transport without affecting the Km for glutamine. Docking of cholesterol into the hASCT2 trimer was performed. Six poses were obtained some of which overlapped the hypothetical cholesterol molecules observed in the available 3D structures. Additional poses were docked close to CARC/CRAC motifs (Cholesterol Recognition/interaction Amino acid Consensus sequence). To test the direct binding of cholesterol to the protein, a strategy based on the specific targeting of tryptophan and cysteine residues located in the neighborhood of cholesterol poses was employed. On the one hand, cholesterol binding was impaired by modification of tryptophan residues by the Koshland's reagent. On the other hand, the presence of cholesterol impaired the interaction of thiol reagents with the protein. Altogether, these results confirmed that cholesterol molecules interacted with the protein in correspondence of the poses predicted by the docking analysis.

Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity

Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15kDa and 5kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4–9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10−3M and 2.8×10−3M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry.

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W102) in the activity of the lectin.

Single Tryptophan of Disordered Loop from Plasmodium falciparum Purine Nucleoside Phosphorylase: Involvement in Catalysis and Microenvironment

Among various tropical diseases, malaria is a major life-threatening disease caused by Plasmodium parasite. Plasmodium falciparum is responsible for the deadliest form of malaria, so-called cerebral malaria. Purine nucleoside phosphorylase from P. falciparum is a homohexamer containing single tryptophan residue per subunit that accepts inosine and guanosine but not adenosine for its activity. This enzyme has been exploited as drug target against malaria disease. It is important to draw together significant knowledge about inherent properties of this enzyme which will be helpful in better understanding of this drug target. The enzyme shows disorder to order transition during catalysis. The single tryptophan residue residing in conserved region of transition loop is present in purine nucleoside phosphorylases throughout the Plasmodium genus. This active site loop motif is conserved among nucleoside phosphorylases from apicomplexan parasites. Modification of tryptophan residue by N-bromosuccinamide resulted in complete loss of activity showing its importance in catalysis. Inosine was not able to protect enzyme against N-bromosuccinamide modification. Extrinsic fluorescence studies revealed that tryptophan might not be involved in substrate binding. The tryptophan residue localised in electronegative environment showed collisional and static quenching in the presence of quenchers of different polarities.

Involvement of tryptophan in the structural alterations of the rat ovarian LH/hCG receptor.

Treatment of the rat ovarian membrane-bound and Triton X-100 solubilized LH/hCG receptor with the tryptophan-specific reagents N-bromosuccinimide (NBS) and 2-hydroxy-5-nitrobenzyl bromide (HNB-Br) resulted in inactivation of the receptor to bind hCG. Fluorescence quenching studies indicated that oxidation of tryptophan residues by NBS decreased the accessibility of fluorophores for acrylamide. Preceding binding of hCG to receptor sites was found to protect fluorophores from NBS action. Modification of tryptophan residues was associated with alteration in the rigidity of ovarian membranes and with destabilization of the LH/hCG receptor structure. The results suggest that tryptophan residue is essential for hCG binding to the receptor.

INN-toxin, a highly lethal peptide from the venom of Indian cobra ( Naja naja) venom—Isolation, characterization and pharmacological actions

A novel toxic polypeptide, INN-toxin, is purified from the venom of Naja naja using combination of gel-permeation and ion-exchange chromatography. It has a molecular mass of 6951.6 Da as determined by MALDI-TOF/MS and the N-terminal sequence of LKXNKLVPLF. It showed both neurotoxic as well as cytotoxic activities. INN-toxin is lethal to mice with a LD 50 of 1.2 mg/kg body weight. IgY raised in chicks against basic peptide pool neutralized the toxicity of INN-toxin. INN-toxin did not inhibit cholinesterase activity. It is toxic to Ehrlich ascites tumor (EAT) cells, but it is not toxic to leukocyte culture. The toxin appears to be specific in its mode of action. Interaction of N-bromosuccinamide (NBS) with the peptide resulted in the modification of tryptophan residues and loss of lethal toxicity of INN-toxin.

Hemagglutinating activity and conformation of a lactose-binding lectin from mushroom Agrocybe cylindracea

A lactose-binding lectin ( Agrocybe cylindracea Lectin, ACL) purified from fruiting bodies of the mushroom A. cylindracea was investigated to determine the hemagglutinating activity and conformation changes after chemical modification, removal of metal ion and treatment at different temperatures and pH. ACL agglutinated both rabbit and human erythrocytes and its hemagglutinating activity could be inhibited by lactose. This lectin was stable in the pH range of 6–9 and temperature up to 60 °C. Fluorescence quenching and modification of tryptophan residues indicated that there were about two tryptophan residues in ACL molecule and one of them might be located on the surface, while the other was buried in the hydrophobic shallow groove near the surface. Chemical modification of serine/threonine and histidine showed that the partial necessity of these residues for the hemagglutinating activity of ACL. However, modifications of arginine, tyrosine and cysteine residues had no effect on its agglutinating activity.

Role of oxidizing mediators and tryptophan 172 in the decoloration of industrial dyes by the versatile peroxidase from Bjerkandera adusta

A purified preparation of the versatile peroxidase from Bjerkandera adusta was tested for industrial dye decoloration. This ligninolytic enzyme efficiently oxidizes Mn(II) to Mn(III) and also exhibits Mn(II)-independent activity in the oxidation of aromatic substrates. It was able to decolorize 27 of the 41 industrial dyes tested. The presence of manganese in the reaction mixture did not enhance the decoloration rate, in fact for some dyes the transformation rate was inhibited. On the other hand, the presence of mediator molecules in the reaction mixture generally enhanced the decoloration of dyes that showed low activity without mediators, while the dyes that were decolorized rapidly without mediator were unaffected by the presence of veratryl alcohol, acetosyringone or TEMPO as oxidizing mediators. The role of tryptophan 172 on dye decoloration was investigated. Chemical modification of tryptophan residues using N-bromosuccinimide drastically reduced Mn(II)-independent activity and dye decoloration, while the Mn(II)-dependent activity was maintained. The loss of Mn(II)-independent and decoloration activities after tryptophan modification was not reversed by the addition of mediators. These results strongly suggest that the tryptophan 172 is involved in both mediator oxidation and dye decoloration activities, and in Mn(II)-independent activity.

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.

Peroxynitrite Inactivates Carbonic Anhydrase II by Releasing Active Site Zinc Ion

Peroxynitrite enters erythrocytes through band 3 anion exchanger and oxidizes cytosolic proteins therein. As a protein associated with band 3, carbonic anhydrase II may suffer from peroxynitrite-induced oxidative damages. Esterase activity of carbonic anhydrase II decreased as the concentration of peroxynitrite increased. Neither hydrogen peroxide nor hypochlorite affected the enzyme activity. Inactivation of the enzyme was in parallel with the release of zinc ion, which is a component of the enzyme's active site. SDS-PAGE of peroxynitrite-treated samples showed no indication of fragmentation but non-denaturing PAGE exhibited new bands with lower positive charges. Western analysis demonstrated that nitration of tyrosine residues increased with the peroxynitrite concentration but the sites of nitration could not be determined. Instead MALDI-TOF analysis identified tryptophan-245 as a site of nitration. Such modification of tryptophan residues is responsible for the decrease in tryptophan fluorescence. These results demonstrate that peroxynitrite nitrates tyrosine and tryptophan residues of carbonic anhydrase II without causing fragmentation or dimerization. The peroxynitrite-induced inactivation of the enzyme is primarily due to the release of zinc ion in the enzyme's active site.

The role of aromatic and acidic amino acids in the electron transfer reaction catalyzed by spinach ferredoxin-dependent glutamate synthase

Treatment of the ferredoxin-dependent, spinach glutamate synthase with N-bromosuccinimide (NBS) modifies 2 mol of tryptophan residues per mol of enzyme, without detectable modification of other amino acids, and inhibits enzyme activity by 85% with either reduced ferredoxin or reduced methyl viologen serving as the source of electrons. The inhibition of ferredoxin-dependent activity resulting from NBS treatment arises entirely from a decrease in the turnover number. Complex formation of glutamate synthase with ferredoxin prevented both the modification of tryptophan residues by NBS and inhibition of the enzyme. NBS treatment had no effect on the secondary structure of the enzyme, did not affect the K ms for 2-oxoglutarate and glutamine, did not affect the midpoint potentials of the enzyme's prosthetic groups and did not decrease the ability of the enzyme to bind ferredoxin. It thus appears that the ferredoxin-binding site(s) of glutamate synthase contains at least one, and possibly two, tryptophans. Replacement of either phenylalanine at position 65, in the ferredoxin from the cyanobacterium Anabaena PCC 7120, with a non-aromatic amino acid, or replacement of the glutamate at ferredoxin position 94, decreased the turnover number compared to that observed with wild-type Anabaena ferredoxin. The effect of the change at position 65 was quite modest compared to that at position 94, suggesting that an aromatic amino acid is not absolutely essential at position 65, but that glutamate 94 is essential for optimal electron transfer.

The essential tryptophan residues of pig kidney aminoacylase.

The tryptophan residues in pig kidney aminoacylase (N-acylamino acid amido hydrolase, EC 3.5.1.14) have been modified by N-bromosuccinimide (NBS) at low pH. The modification of eight tryptophan residues as measured by spectrophotometric and spectrofluorimetric methods leads to complete loss of enzymatic activity. The decreases in absorption at 280 nm and fluorescence emission at 337 nm indicate the modification of tryptophan residues. Both the inactivation and tryptophan residual modification are monophasic, first-order reactions. Quantitative treatment of the data (Tsou, C. L., Sci. Sin., 1962, 11, 1535-1558) shows that among the tryptophan residues modified, two are essential for aminoacylase catalytic activity. Kördel and Schneider (Hoppe-Seyler's Physiol. Chem. 1976, 357, 1109-1115) reported that the modification of tryptophan residues led to inactivation of aminoacylase, and suggested that tryptophan residues are essential for enzymatic activity. We have now shown that eight tryptophan residues can be modified by N-bromosuccinimide and that two of them are essential for the catalytic activity of this enzyme.

Characterization of cloned endoxylanase from Cellulomonas sp. NCIM 2353 expressed in Escherichia coli.

A 22-kDa xylanase encoded by a cloned gene (XCs16)of Cellulomonas was purified to homogeneity with an overall yield of 44%. It is a basic protein with a pI of 8.1 and has a Km and Vmax of 3 mg/ml and 1150 micromoles/mg/min, respectively, for oat spelt xylan at 55 degrees C and pH 5.8. Homologous xylanase from Cellulomonas could be identified with antibodies raised against purified xylanase encoded by XCs16. The enzyme from Cellulomonas also exhibited identical temperature and pH optimum and had a molecular weight of 23 kDa. Modification of tryptophan residue of purified xylanase resulted in the loss of xylanase activity. This loss could be reversed by the addition of substrate, indicating the involvement of tryptophan residue in the catalytic site.

An essential tryptophan residue of green crab (Syclla Serrata) alkaline phosphatase

The tryptophan residues in green crab (scylla serrata) alkaline phosphatase (EC 3.1.3.1) have been modified by N-bromosuccinimide (NBS). The modification of five tryptophan residues leads to complete loss of enzymatic activity. With the increase of NBS concentration, both the absorption at 278 nm and the fluorescence emission intensity at 335 nm of the modified enzyme decreased markedly indicating the modification of tryptophan residues. Quantitative treatment of the data (Tsou, Sci. Sinica 1962, 11, 1535-1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of the substrate markedly protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.

Chemical Modification and Inactivation of Rat Liver Arginase byN-Bromosuccinimide: Reaction with His141

Treatment of rat liver arginase withN-bromosuccinimide results in modification of six tryptophan residues per enzyme molecule and is accompanied by loss of catalytic activity (E. Ber and G. Muzyńska (1979)Acta Biochim. Pol.26, 103–114). In order to probe the chemistry ofN-bromosuccinimide inactivation and the role of tryptophan residues in catalysis, the two tryptophan residues of rat liver arginase, Trp122 and Trp164, have been separately mutated to phenylalanine using site-directed mutagenesis of the protein expressed inEscherichia coli.Both single Trp→Phe mutant enzymes have kinetic parameters nearly identical to those for the wild-type enzyme. Treatment of native, wild-type, and each of the Trp→Phe mutant enzymes withN-bromosuccinimide results in loss of absorbance at 280 nm and is accompanied by a loss of catalytic activity. However, treatment of the wild-type enzyme withN-bromosuccinimide in the presence of the arginase inhibitorsNG-hydroxy-L-arginine or the combination ofL-ornithine and borate protects against inactivation, even though tryptophan residues are modified. Treatment of the H101N and H126N mutant arginases withN-bromosuccinimide also results in loss of catalytic activity and modification of tryptophan residues. In contrast, the H141N mutant arginase is not inactivated byN-bromosuccinimide, indicating that His141 is the critical target for theN-bromosuccinimide inactivation of the enzyme.

Leader peptidase from Escherichia coli: overexpression, characterization, and inactivation by modification of tryptophan residues 300 and 310 with N-bromosuccinimide.

We have overproduced the leader peptidase from Escherichia coli in a high yield by using a T7 RNA polymerase/promoter system and purified the enzyme. This leader peptidase showed an apparent pH optimum of about 10 toward a synthetic peptide substrate, and was stable at temperatures below 40 degrees C. Kinetic studies indicated that one of the active site residues in the enzyme has a pKa value of approximately 7.5. The enzyme was rapidly inactivated by reaction with N-bromosuccinimide (NBS). When approximately two tryptophan residues were oxidized with NBS, the activity was almost completely lost and this inactivation was markedly prevented by a substrate. These NBS-reactive tryptophan residues were identified as Trp300 and Trp310 by a peptide mapping analysis. This indicates that Trp300 and/or Trp310 are critically important for the activity of the leader peptidase. On the other hand, the enzyme was scarcely inhibited by treatment with N-acetylimidazole, iodoacetic acid, 5,5'-dithiobis(2-nitrobenzoic acid), succinic anhydride, or 2,4,6-trinitrobenzenesulfonate. Diethylpyrocarbonate inhibited the enzyme; however, this inhibition did not seem to result from the modification of histidine residues. Thus, there seem to be no functionally important tyrosine, cysteine, or histidine residues or amino groups among the residues which readily react with these reagents. However, the enzyme was inactivated significantly by treatment with phenylglyoxal or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Therefore, some of the arginine residues and the carboxyl groups appear to be important for the enzyme activity.

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 a...

Cycloamylose Glucanotransferase‐catalyzed Cyclisation for a Substrate Maltose. Modification with N‐Bromosuccimide on the Tryptophan Residues

AbstractCycloamylose glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus stearothermophilus was found to catalyze the production of cyclomalto‐oligosaccharides from maltose (G2) as a substrate. This synthesis reaction, called “cyclisation” is one of the four CGTase‐catalyzed reactions: disproportionation, coupling, hydrolysis, and cyclisation. On the reaction time‐course observed by using the fluorescence method, a lag‐phase was found prior to the production of cyclomalto‐oligosaccharides. The lag‐time may be referred to a reaction “disproportionation”.Based on the linear portion of the reaction curves, the kinetic parameters, the Michaelis constant Km and the molar activity k0, were evaluated for the G2 cyclisation. Modification of tryptophan residues with N‐bromosuccinimide gives not so much effect on the Km and k0 values for cyclisation. However, an effect was found on the lag‐time, suggesting that the Trp residue (Trp97) carries an essential role in the disproportionation process.

Tryptophan Residues of Bacillus Cycloamylose Glucanotransferase: Effect of Modification with N‐Bromosuccini‐mide on the Enzyme‐catalysed Synthesis of Cyclomaltoheptaose from Maltotriose

AbstractCycloamylose glucanotransferase(cyclomalto‐oligosaccharide glu‐canotransferase, CGTase), obtained from Bacillus stearotheromophilus, was found to catalyze the synthesis of cyclomalto‐oligosaccharides from maltotriose (G3) as a substrate. The synthetic reaction is one of the CGTase‐catalysed reactions, so called “cyclisation”. A main saccharide produced by the cyclisation reaction under the experimental conditions employed was confirmed to be cyclomaltoheptaose CG7.The kinetic parameters, the Michaelis constant Km and the molar activity k0 were evaluated for the cyclisation reaction. Modification of tryptophan residues with N‐bromosuccinimide (NBS) was found to effect on Km, but not on k0 of the cyclisation reaction, suggesting that the tryptophan residue (Trp97) carries an essential role in the binding (disproportionation process) of maltotriose.