Substrate size dependence of lysozyme-catalyzed reaction

“…In the calculation of the time-course, the rate constant value of k +1 (for cleavage of the glycosidic linkage) was assumed to be dependent upon the substrate size (13).…”

Section: Methodsmentioning

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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“…A kinetic model for the lysozyme-catalyzed reaction of chito-oligosaccharides has been reported, [31][32][33] and is presented in a simplified form by omitting some details of the reaction (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

“…[31][32][33] We can therefore directly evaluate the contributions of amino acid substitution at the active site to substrate binding and enzymatic reactions by combining the results of an HPLC analysis of the reaction products and theoretical calculation of the reaction timecourse data. 10,11,[34][35][36][37][38] The naturally occurring lysozyme molecules closely and genetically related to HEL, which carry limited amino acid substitutions at the substrate binding site, are useful to evaluate the reaction mechanisms of lysozyme such as substrate binding and lysozyme catalysis.…”

mentioning

confidence: 99%

“…The time-course characteristics of the EGL-catalyzed reaction on the GlcNAc pentamer substrate, (GlcNAc) 5 , were measured for comparison with those of HEL, and the binding constants of sugar residues at each subsite and the rate constants were estimated from the experimental time-course data through computer simulation of the lysozyme-catalyzed reaction. [31][32][33] Site-specific mutations of residues 34, 37 and 71 in HEL were also conducted, and the mutated proteins (F34Y, N37G, F34Y/N37G, G71R, and F34Y/N37G/G71R) were subjected to activity measurements. We found Tyr34, Gly37, and Arg71 in EGL to be the key residues responsible for the time-course characteristics of EGL.…”

mentioning

confidence: 99%

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