Sequence specificities for lysozyme depolymerization of partially N-acetylated chitosans

Abstract

The influence of sugar residue sequence in partially N-acetylated chitosans on relative hydrolysis rate catabzed by lysozyme was studied. The relative rates were modelled assuming an Arrhenius-type relation for the relative rate constants. The apparent activation energy was assumed to consist of additive contributions from GlcN or GlcNAc residues within the polymer chain interacting with sites A-F of the active cleft of lysozyme. This model accounted well for the relative hydrolysis rates reported for well-defined oligomers. Calculated and experimental data for the dependence of the initial relative hydrolysis rates on fraction of acetylated units, FA, showed an F~~.~ dependence. A fully water-soluble highly N-acetylated chitosan with FA = 0.68 was depolymerized using lysozyme for further testing of the model. Analyses of the I3c nuclear magnetic resonance spectra of the diad sequences at the new reducing and nonreducing ends formed by lysozyme showed that this enzymatic depolymerization was dominated by chitosan sequences presenting GlcNAc residues to sites C, D, and E of the active cleft. In contrast, there was no selectivity between GlcNAc and GlcN residues interacting with site F. These selectivities were confirmed by the calculated contributions to the apparent activation energy of these sites. The experimentally determined depletion in the diad and triad frequencies of GlcNAc during the course of lysozyme hydrolysis was in good agreement with the model calculations.Key words: lysozyme, chitosan, chitin, sequence specificity, subsite model.Resume : On a CtudiC l'influence de la sCquence des rtsidus de sucre, dans des chitosanes partiellement N-acCtylCs, sur le vitesses relatives d'hydrolyse par le lysozyme. On a fait un modkle des vitesses relatives en supposant qu'on peut leur appliquer une relation du type d'Arrhenius. On a supposC que 1'Cnergie d'activation apparente est formie de contributions additives des rCsidus GlcN ou GlcNAc prksents dans la chai'ne de polymkre avec les sites A 2i F de la cavitC du lysozyme. Ce modkle a permis d'expliquer les vitesses d'hydrolyse relatives rapportCes pour des oligomkres bien dCfinis. Les donnCes calculCes et expCrimentales concernant la dtpendance des vitesses d'hydrolyse relatives initiales sur la fraction d'unitts acCtylCes, FA, prtsentent une dtpendance Dans le but de vCrifier plus a fond le modble et utilisant le lysozyme, on a dCpolymCrisC un chitosane N-acCtylC, complktement soluble dans l'eau et de FA = 0,68. Des analyses par de la rCsonance magnttique nuclCaire I3c des sequences de diades aux extrtmitCs rkductrices et non rtductrices formCes par le lysozyme ont montrC que cette dCpolymCrisation enzymatique est dominCe par des sCquences de chitosane prksentant des rtsidus GlcNAc aux sites C, D et E de la cavitt active. Par opposition, aucune sClectivitC a pu Etre observCe entre les rCsidus GlcNAc et GlcN qui interagissent avec le site F. On a confirm6 ces sClectivitCs par les contributions calculCes a I'Cnergie apparente d'activation de ces sites. ...

The Recombinant Azotobacter vinelandii Mannuronan C-5-Epimerase AlgE4 Epimerizes Alginate by a Nonrandom Attack Mechanism

“…Although the copolymer nature of the substrate indicates that several types of substrates coexist in the alginate chains, this two-substrate approximation is the simplest extension over the one-substrate model that can be realized. The preferred attack model was implemented using a Monte Carlo scheme (43,44) on the basis of alginate chains with a degree of polymerization (DP n ) of 30, as above.…”

Direct Determination of Chitosan–Mucin Interactions Using a Single-Molecule Strategy: Comparison to Alginate–Mucin Interactions

“…The enzymatic processing of chitosan, including degradation and de-acetylation, is mediated by dynamic realization of a set of interactions, in particular hydrogen bonds, between molecular groups of the chitosan and the enzyme [6,7]. Enzymatic catalysis depends on chitosan-enzyme interactions spanning a set of chitosan residues interacting with the sub-sites of the catalytic cleft, thus producing a reaction rate that reflects the local sequence of the polysaccharide [8,9].…”

Development and application of a model for chitosan hydrolysis by a family 18 chitinase

“…14 - 24 Allen and Thoma 20 have described a procedure for analyzing the interactions between an enzyme and its polymeric substrate, which is referred to as subsite mapping. In this approach, steady-state kinetic parameters for a series of oligosacharides need to be measured and information on the chain-lengths of the hydrolysis products needs to be acquired.…”

“…Stokke et al 24 used an approach similar to that of Allen and Thoma 20,21 to study the sequence specificities for depolymerization of chitosan by a nonprocessive enzyme (lysozyme). Relative hydrolysis rates of various tri-and tetrasaccharides were used to create a subsite model.…”

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