Kinetics of Chitinase from Yam,<i>Dioscorea opposita</i>THUNB

Koga, Daizo; Tsukamoto, Tsuyoshi; Sueshige, Nobuyuki; Utsumi, Toshihiko; Ide, Akio

doi:10.1080/00021369.1989.10869822

Cited by 5 publications

(7 citation statements)

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“…Similar profiles were obtained, when (GlcNAc) 2 -4MU and (GlcNAc) 3 -4MU were used as the substrate (Figure 4b). The use of such substrates, which release a fluorescent product, is a common and simple assay for chitinase activity (25,26). When used as substrates for class 19 chitinases, these artificial substrates exhibit hyperbolic kinetics for which K m and k cat values can be readily measured (26).…”

Section: Discussionmentioning

confidence: 99%

Kinetic Properties of Chitinase-1 from the Fungal Pathogen Coccidioides immitis

et al. 2001

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The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the -conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc) 6 is predominantly cleaved into (GlcNAc) 2 and (GlcNAc) 4 , where the (GlcNAc) 2 group arises from the nonreducing end of the substrate and is formed as the -anomer. With time, transglycosylation occurs, generating (GlcNAc) 8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc) 5 and (GlcNAc) 4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc) 4 is 50 µM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc) 6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.

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Section: Discussionmentioning

confidence: 99%

Kinetic Properties of Chitinase-1 from the Fungal Pathogen Coccidioides immitis

et al. 2001

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“…Koga and co-workers used di through hexa NAG substrates to carry out a kinetic analysis of the yam chitinase (Koga et al 1989). They showed k, , , was no more than 501min for tetraNAG but only 0. llmin for triNAG.…”

Section: Tyr123mentioning

confidence: 99%

The structure of chitinases and prospects for structure-based drug design

Robertus¹,

Hart²,

Monzingo³

et al. 1995

Can. J. Bot.

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Many fungi, including pathogenic strains, require proper chitin metabolism to assure normal cell wall replication. Chitinase hydrolyzes chitin; inhibition of endogenous chitinases or application of extracellular chitinases can disrupt fungal division. It is possible that chitinase inhibitors could be used as antifungal agents. We have solved the X-ray structure of a class I1 chitinase from barley and proposed a mechanism of action. The enzyme has a structural core similar to lysozyme and probably acts in a similar catalytic manner. The enzyme structure can, in principle, be used to identify small molecules that will bind avidly to the active site and act as inhibitors. Those inhibitors that embody transition state geometry are likely to be particularly effective.Key words: chitinase, mechanism of action, drug design.RCsumk : Plusieurs champignons, incluant des souches pathogtnes, ont besoin d'un mttabolisme de la chitine approprit pour assurer une reproduction normale de la paroi cellulaire. La chitinase hydrolyse la chitine; l'inhibition des chitinases endogknes ou l'application de chitinases extracellulaires peut bloquer la division fongique. I1 est possible que des inhibiteurs de la chitinase puissent Ctre utilists comme substances antifongiques. Les auteurs ont dtchiffrt la structure, aux rayons X, d'une chitinase de classe I1 chez I'orge, et proposent un mtcanisme d'action. L'enzyme posstde une structure centrale semblable i celle du lyzozyme et agit probablement d'une fa~on catalytique comparable. La structure de l'enzyme peut, en principe, Ctre utiliste pour identifier de petites moltcules qui s'attachent fortement au site actif pour agir comme inhibiteurs. Ces inhibiteurs, qui montrent un ttat gtomttrique de transition, ont des chances d'Ctre particulikrement efficaces.

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“…In contrast, insects have a very substrate-specific chitinase, which is consistent with the hypothesis that they use it only in ecdysis. 20) Chitinase from Streptomyces griseus 29 ) mainly splits GlcNAc 2 from the non-reducing end of p-nitrophenylchitooligosaccharides, and releases p-nitrophenol from pNp-GlcNAc2' The chitosanolytic enzyme from Enterobacter sp. G-I that we isolated also mainly released GlcNAc 2 from the non-reducing end of N-acetylchitooligosaccharides and p-nitophenyl-chitooligosaccharides.…”

Section: Discussionmentioning

confidence: 99%