Resolution of the individual steps in the reaction of lysozyme with the trimer and hexamer of N-acetyl-D-glucosamine at subzero temperatures

Abstract

The reaction of hen egg white lysozyme with chitotriose and chitohexose has been investigated at temperatures to below -100 degrees C by using aqueous methanol and dimethyl sulfoxide cryosolvents. Initial investigations involving the effects of increasing cosolvent concentration on the catalytic and structural properties of the enzyme indicated that both methanol and dimethyl sulfoxide cryosolvents, at subzero temperatures, had no adverse effects on lysozyme. Time-dependent changes in the fluorescence emission of the enzyme, under nonturnover conditions, permitted the detection of two intermediates in the reaction with the trimer and three intermediates in the case of the hexamer. The fastest reaction observed for both substrates was complete within minutes at -90 degrees C and is attributed to initial substrate binding, followed by rapid isomerization to form a "loose" ES complex. The subsequent, slower reactions correspond to successive isomerizations of ES to "tighter" complexes. From dye binding displacement reactions, and comparison of dissociation constants, it is concluded that the predominant mode of binding for the hexamer at subzero temperature is productive. Rate and dissociated constants were determined for the observed reactions as a function of temperature, pH, and cosolvent concentration. Overall the reaction pathway in methanol cryosolvents at subzero temperatures appears to be very similar to that determined from investigations in aqueous solution at ambient temperatures using a variety of rapid reaction techniques. Conditions necessary to accumulate and stabilize each of the observed intermediates have been determined, thus permitting further studies to delve more deeply into the nature of the intermediates.

Experimental identification of a theoretically predicted "left-sided" binding mode for (GlcNAc)6 in the active site of lysozyme

“…Previous studies (Smith-Gill et al, 1982) have already shown that the dye Biebrich Scarlet [known to bind exclusively to the F site of HEWL (Holler et al, 1975)] is displaced by HyHEL-5 from HEWL, indicating that the dye itself lies at least partially in the region of residues 45-47. In Figure 1, the results of competition experiments are shown in which different oligosaccharides of GlcNAc are allowed to compete with HyHEL-5 in binding to HEWL. These experiments were conducted at -20 °C, at which temperature no catalysis occurs but binding is actually stronger (Douzou et al, 1974;Fink et al, 1980).…”

“…Substrate inhibition of antibody binding to HEWL was examined in a low-temperature ELISA plate-binding assay. It is known from previous work (Douzou et al, 1974;Fink et al, 1980) that, at temperatures below -20 °C in a variety of antifreeze buffers, the rate of lysozyme-catalyzed hydrolysis approaches zero (i.e., kcat = 0) for cell wall substrates and for (GlcNAc)6. However, the binding of substrates to the enzyme is actually enhanced (Douzou et al, 1974;Fink et al, 1980).…”

“…It is known from previous work (Douzou et al, 1974;Fink et al, 1980) that, at temperatures below -20 °C in a variety of antifreeze buffers, the rate of lysozyme-catalyzed hydrolysis approaches zero (i.e., kcat = 0) for cell wall substrates and for (GlcNAc)6. However, the binding of substrates to the enzyme is actually enhanced (Douzou et al, 1974;Fink et al, 1980). Furthermore, the fact that Arrhenius plots of the logarithm of the rate of reaction vs. temperature over all temperatures between 25 and -20 °C are linear suggests that no major structural changes occur in the enzyme-substrate complex (Douzou et al, 1974;Fink et al, 1980).…”

“…However, the binding of substrates to the enzyme is actually enhanced (Douzou et al, 1974;Fink et al, 1980). Furthermore, the fact that Arrhenius plots of the logarithm of the rate of reaction vs. temperature over all temperatures between 25 and -20 °C are linear suggests that no major structural changes occur in the enzyme-substrate complex (Douzou et al, 1974;Fink et al, 1980). In the competitive binding experiments, 96-well V-bottom microtiter plates were coated with 50 nL of 1 jig/mL HEWL for 1 h followed by 1% BSA for 1 h at room temperature and then frozen overnight at -22 °C.…”

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

“…1). Considerable efforts have previously been made, with HEWL, to ®nd such a condition yet in no case have these efforts proven successful 9,10 . This observation strongly suggests that, for HEWL, hydrolysis of the intermediate (k 3 ) occurs at a much greater rate than does its formation (k 2 ).…”