Estimation of rate constants in lysozyme-catalyzed reaction of chitooligosaccharides.

The rate constants of the cleavage of glycoside linkage, hydration (hydrolysis) and transglycosylation in a lysozyme-catalyzed reaction of substrate chitooligosaccharides were evaluated by computer analysis of the experimentally obtained reaction time-courses. In the computer analysis, the rate equation was numerically solved by use of the known binding constants for each subsite. Because of the complexity of the lysozyme-catalyzed reaction, optimal values of rate constants were determined by checking the sensitivity of each rate constant to the computed time-courses. It was not possible to estimate uniquely the rate constants for transglycosylation and hydration, owing to the nature of the enzymatic reaction, but it was possible to estimate accurately their ratio. The estimated values were 0.94 s-1 for the rate constant for the cleavage of glycosidic linkage and 133 for the ratio of rate constants of transglycosylation and hydration.

Lysozyme-catalyzed reaction of chitooligosaccharides.

The time-courses of substrate consumption and product formation in the lysozyme-catalyzed reaction were determined with (GlcNAc)4 and (GlcNAc)5 as substrate to accumulate data suitable for the estimation of rate constants by numerical analysis. The lysozyme-catalyzed reactions were followed by TLC or HPLC. (GlcNAc)4 decomposed apparently to small oligosaccharides within 5 h, and (GlcNAc)5 decomposed within 15 min at pH 5.0 and 50 degrees C. The temperature-dependence of the rate of disappearance of the initial substrate showed a different profile from that observed with glycol chitin as substrate by the reducing power method. The order (or distribution) of the amount of product formed from (GlcNAc)5 in the reaction time-course determined by TLC differed from that determined by HPLC. The relative error in HPLC was much less than that in TLC, and the time-course determined by HPLC was thought to be of sufficient accuracy for the estimation of rate constants by computer analysis.