Lysozyme hydrated, enzymatic activity

Fig. 29. Enzymatic activity of lysozyme as a function of water content (grams of water per gram of sample), at pH 8, 9, and 10. , O, A, Measurements on powders hydrated by isopiestic equilibration ,, A, solvent added to powder. Powder samples were the 1 1 (GlcNAc)6-lysozyme complex, obtained by lyophilization. The reaction rate (no sec ) was determined by product analysis. From Rupley etcd. (1980).

The effect of "residual water" on either protein stability or enzyme activity continues to be a topic of great interest. For example, several properties of lysozyme (e.g., heat capacity, diamagnetic susceptibility (Hageman, 1988), and dielectric behavior (Bone and Pethig, 1985 Bone, 1996)) show an inflection point at the hydration limit. Detailed studies on the direct current protonic conductivity of lysozyme powders at various levels of hydration have suggested that the onset of hydration-induced protonic conduction (and quite possibly for the onset of enzymatic activity) occurs at the hydration limit. It was hypothesized that this threshold corresponds to the formation of a percolation network of absorbed water molecules on the surface of the protein (Careri et al., 1988). More recently. Smith et al., (2002) have shown that, beyond the hydration limit, the heat of interaction of water with the amorphous solid approaches the heat of condensation of water, as we have shown to be the case for amorphous sugars. 

Figure 7. Enzymatic activity of lysozyme as a function of water content, at pH 8, 9, and 10. Open symbols measurements on powders hydrated by isopestic eqiulibra-tion. Closed symbols solvent added to powder.

Up to a certain extent, enzymes need to be hydrated in order to develop activity. Hydration of e. g. lysozyme was determined by IR and NMR spectroscopy. As can be seen in Table 2.15, first the charged polar groups of the side chains hydrate, followed by the uncharged ones. Enzymatic activity starts at a water content of 0.2 g/g protein, which means even before a monomolec-ular layer of the polar groups with water has taken place. Increase in hydration resulting in... 

At molecular level, the manifestations of the biological activity appear in specific biochemical reactions, conformational behavior, and dynamical properties of biomolecules. Experimental studies of various partially hydrated enzymatic proteins show that their activity accelerates rapidly at some critical hydration levels. Onset of the enzymatic activity of urease occurs at 0.15 g/g [469]. In the presence of chymotrypsin, the acylation reaction is undetectable at hydrations /i< 0.12 g/g, but its rate grows sharply above this critical hydration level [470]. The rate of enzymatic activity of glucose-6-phosphate dehydrogenase, hexoki-nase, and fumarase becomes detectable and start to increase sharply at hK 0.20 g/g, whereas this critical hydration is about 0.15 g/g for phosphoglucose isomerase [471]. Enzymatic activity of lysozyme can be detected only when hydration level achieves h 0.20 g/g [472, 473] (see Fig. 92). 

Figure 92 The rate a of the enzymatic activity of lysozyme at various hydration levels [473].

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