Biomedical subjects

T Fukamizo

Publications and source records attributed to T Fukamizo.

41 records · Page 3Linked to original sources

PubMed1982

Estimation of the free energy change of substrate binding lysozyme-catalyzed reactions.

The binding constant and binding free energy of each subsite of lysozyme upon substrate binding have been customarily estimated from the experimental data with assumptions regarding the binding mode of substrate and the additivity of binding free energy of each subsite. In the present study, the binding constants and binding free energy of subsites were estimated from experimentally obtained overall binding constants on native and Trp 62-modified lysozymes. The estimations of binding constants and binding free energy were carried out by an optimization method, the modified Powell method, without assuming the binding mode for substrate. First the binding free energies of subsites A, B, and C were estimated from the experimental binding constants of (GlcNAc)1 to (GlcNAc)3, and the binding free energies of subsites D, E, and F were determined from the estimated free energies of subsites A, B, and C, and the experimentally obtained reaction time-courses of substrate (GlcNAc)5. Finally, the values of three rate constants in the lysozyme-catalyzed reaction of chitooligosaccharide were estimated from the experimental time-course by using the binding free energies obtained by the modified Powell method.

Animals↗

PubMed1982

Human lysozyme-catalyzed reaction of chitooligosaccharides.

The time-courses of the human lysozyme-catalyzed reaction of chitopentaose were measured by high-performance gel-filtration in comparison with those of hen egg-white lysozyme. Human lysozyme has considerably larger rate constants for the cleavage of glycosidic linkages and transglycosylation than those of hen lysozyme, in agreement with the fact that human lysozyme exhibits a large lytic activity. It has been reported that binding subsite D in human lysozyme has negative free energy on substrate binding, whereas subsite D in hen lysozyme has unfavorable positive free energy due to the distortion of a sugar residue. The time-courses calculated under the assumption that subsite D in human lysozyme has negative free energy on substrate binding did not fit the experimentally obtained time-courses, even though the combination of values of rate constants in the enzymatic reaction widely varied in the calculation of the time-courses. Thus, it was concluded that subsite D in human lysozyme may not have negative binding free energy, but positive values similar to hen lysozyme.

Animals↗

PubMed1982

Enzymatic activity of Trp 62-modified lysozyme.

The time-courses of action of Trp 62-modified lysozymes on the initial substrate chitopentaose were measured by means of high-performance gel-filtration. The activities of the modified lysozymes, represented by the rate of disappearance of the initial substrate (overall rate) were lowered to various extents depending on the method of the modification. On the other hand, the time-courses were calculated by changing the values of rate constants, using the binding free energy of each subsite estimated by the optimization technique (Kuhara et al. (1982) J. Biochem.). For NBS- and NPS-lysozymes, the calculated time-courses were not in good agreement with the experimental ones, when the binding free energies estimated by the optimization technique were used for the calculation. Therefore, the binding free energies of the subsites were estimated from the experimental time-courses with the assumption that the values of the rate constants do not change upon modification of Trp 62 in subsite C. As a result, it was found that, though the modification was at subsite C, the binding free energy of subsite A was profoundly lowered, while that of subsite B remained almost unchanged.

Energy Metabolism↗

PubMed1981

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.

Acetylglucosamine↗

PubMed1981

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.

Acetylglucosamine↗