Resolution of the individual steps in the reaction of lysozyme with the trimer and hexamer of N-acetyl-D-glucosamine at subzero temperatures.
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.
D000117:Acetylglucosamine; D000818:Animals; D002645:Chickens; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004529:Egg White; D005615:Freezing; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics
D009844:Oligosaccharides; D009113:Muramidase; D000117:Acetylglucosamine; D004121:Dimethyl Sulfoxide