Lysozyme hydrolysis rate

Hydrolysis Rate Constants for Model Oligosaccharides with Lysozyme... 

Moreover, initial experiments using a standard protocol suggest that the rate of degradation of PHB in vivo is significantly faster than the in vitro hydrolysis rate at the same temperature and pH. The obvious interpretation is that non-specific esterase and lysozyme enzymes secreted by the body s immune system catalyse the process. Indeed it is relatively easy to demonstrate that the polymer is attacked by hog s liver esterase, for example. 

The opaque chitosan gel was treated with acetic anhydride in an aqueous methanol solution at room temperature overnight, and a transparent gel was obtained. The gel was indistinguishable from chitin gel prepared from chitosan by N-acetylation in aqueous acetic methanol on the basis of its IR and CP/MAS-NMR spectra, on the hydrolysis rate by lysozyme... 

As shown in Table 2, the xerogels prepared by these methods were hydrolyzed at almost the same rate by hen egg-white lysozyme, and the rate was about 6 times higher than that of crab shell chitin. The hydrolysis rate by lysozyme was slightly inhibited by the presence of 0-acetyl group in chitin xerogel, probably because of the steric hindrance of the 0-acetyl group at Ce for forming an enzyme substrate complex. The increase in the rate of enzymic hydrolysis also proves the formation of the same chitin gel by three independent methods. 

The reduction of the rate of hydrolysis due to lowering the temperature 45 C in the solution containing 7 M NH4NO3 is Ah2o/ nh4N03,7m = 4.78, with an activation energy of 5 kcal mol . This solvent is not suitable for low-temperature studies of the lysozyme reaction. 

Fig. 14. The effect of ionic strength on the pH-rate profile of the lysozyme-catalyzed hydrolysis ofM. lysodeikticus cell walls. As ionic strength increases, the pH optimum shifts toward acidic pH values.

The maximum rate is directly related to the rate at which the enzyme processes or permits conversion of the reactant molecule(s). The number of moles of reactants processed per mole of enzyme per second is called the turnover number. Turnover numbers vary widely. Some are high, such as for the scavenging of harmful free radicals by catalase, with a turnover number of about 40 million. Others are small, such as the hydrolysis of bacterial cell walls by the enzyme lysozyme, with a turnover number of about one half. 

Figure 5. Separation of ozonized lysozyme CNBr fragments. The ozonized lysozyme was treated with CNBr as described in the Materials and Methods. The peptide mixture was applied to a column of Sephadex G-25 (3 X 100 cm) and eluted with 0.2N HAc at a flow rate of 0.5 ml/min. Fractions were assayed by measuring UV absorbance on ninhydrin color after alkaline hydrolysis.

Bell-shaped pH-rate profiles are obtained in lysozyme reactions (Rupley et al., 1967) which are consistent with direct involvement of two groups in the reaction. However, bell-shaped pH-rate constant profiles are also observed in the hydrolysis of benzaldehyde disalicyl acetals, and in the case of p-nitrobenzaldehyde o-carboxyphenyl p-carboxyphenyl acetal only one carboxyl group can participate. One should then take care in postulating bifunctional catalysis in the lysozyme reaction, since the observed kinetics and the rate enhancements are explicable in terms of a chemically simpler mechanism (general acid catalysis by glutamic acid-35 along with release of ground state... 

Figure 1. Comparison of the catalytic activity of WT lysozyme and the 7-methionine mutant (see text). The enzymatic hydrolysis of peptidoglycan is followed as a change in circular dichroism. Rates are obtained from the slopes, as indicated (see text for details).

Fig. 24.2. Single-molecule recording of T4 lysozyme conformational motions and enzymatic reaction turnovers of hydrolysis of an E. coli B cell wall in real time, (a) This panel shows a pair of trajectories from a fluorescence donor tetramethyl-rhodamine blue) and acceptor Texas Red (red) pair in a single-T4 lysozyme in the presence of E. coli cells of 2.5mg/mL at pH 7.2 buffer. Anticorrelated fluctuation features are evident. (b) The correlation functions (C (t)) of donor ( A/a (0) Aid (f)), blue), acceptor ((A/a (0) A/a (t)), red), and donor-acceptor cross-correlation function ((A/d (0) A/d (t)), black), deduced from the single-molecule trajectories in (a). They are fitted with the same decay rate constant of 180 40s. A long decay component of 10 2s is also evident in each autocorrelation function. The first data point (not shown) of each correlation function contains the contribution from the measurement noise and fluctuations faster than the time resolution. The correlation functions are normalized, and the (A/a (0) A/a (t)) is presented with a shift on the y axis to enhance the view, (c) A pair of fluorescence trajectories from a donor (blue) and acceptor (red) pair in a T4 lysozyme protein without substrates present. The acceptor was photo-bleached at about 8.5 s. (d) The correlation functions (C(t)) of donor ((A/d (0) A/d (t)), blue), acceptor ((A/a (0) A/a (t)), red) derived from the trajectories in (c). The autocorrelation function only shows a spike at t = 0 and drops to zero at t > 0, which indicates that only uncorrelated measurement noise and fluctuation faster than the time resolution recorded (Adapted with permission from [12]. Copyright 2003 American Chemical Society)...

Enzymic resolutions involve acceptance by the enzyme, which is a very finely honed chiral system, of one enantiomer of a racemic compound, but not the other. The selective acceptance arises because interactions between the enzyme and the enantiomers are diastereomeric. In its natural environment, the ability of an enzyme to discriminate between enantiomers is virtually absolute. In addition to their stereoselectivity, some enzymes can react at very high rates. Each round of catalysis by the enzyme carbonic anhydrase with its physiological substrate occurs in about 1.7 jus at room temperature, although for a small number of other enzymes, best exemplified by the more lethargic lysozyme, the corresponding figure is about a million times slower. Accordingly, the enzyme-catalysed hydrolysis of, say, one enantiomer of an ester proceeds at a finite rate and hydrolysis of the other not at all. Resolutions such as those of 39, 42 and 45 therefore have a kinetic basis and are also known as kinetic resolutions. 

Dialysis and recrystallisation are simple and yield enzyme of high purity. Several forms of crystals are obtained depending on the pH of the crystallising solution. The activity of lysozyme is measured by the rate of decrease of turbidity (at 570nm ) as hydrolysis of acetone-dried cell walls of the Gram-positive bacterium Micrococcus lysodeikitus (as substrate) occurs on addition of the enzyme [Hirs Methods Enzymol I 124 1968]. 

An important question regarding peptides and proteins is concerned with the equilibria among several conformational states. It has been suggested that enzyme function may be linked to the occurrence of particular conformations in solution.24 377 A mechanism recently proposed for the hydrolysis of oligosa-charides by the enzyme lysozyme, for example, is based on the observation of specific substrate and enzyme sidechain conformations in a molecular dynamics simulation of a lysozyme-substrate complex.378 Also, local conformational equilibria and the barriers between conformations are important in determining the rates and mechanisms of folding and rebinding processes. 

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