HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sophianopoulos, A. J. Search for Related Content PubMed PubMed Citation Articles by Sophianopoulos, A. J. Social Bookmarking                      What's this?

Association Sites of Lysozyme in Solution

I. THE ACTIVE SITE

From the ¹ From the Department of Biochemistry, Emory University, Atlanta, Georgia 30322

Sedimentation equilibrium experiments with lysozyme at 13° and 20°, 0.15 ionic strength, and near pH 8 have shown that the presence of N-acetylglucosamine and of the disaccharide (GlcNAc-GlcNAc) and trisaccharide (GlcNAc-GlcNAc-GlcNAc) greatly reduces the extent of reversible dimerization of lysozyme reported previously (Sophianopoulos, A. J., and Van Holde, K. E., J. Biol. Chem., 239, 2516 (1964)). This indicates that the enzymatically active region of lysozyme is one of the sites of contact for the formation of dimer in solution or is near such a site. The dimer dissociation constant of lysozyme in the absence of saccharide at 20° and under the conditions described above is K_d = (P)²/(PP) = (2.71 ± 0.24) x 10^-3 m. If it is assumed that saccharide (S) can bind to the monomer protein species (P) to form a complex (PS) but that the dimer species (PS·PS) cannot be formed, the values for the dissociation constant of the PS complex K_s = (P)(S)/(PS) can be calculated indirectly, by using exclusively results from sedimentation equilibrium experiments in the presence and absence of the saccharides. Two distinct sets of values of K_s are obtained, depending on whether it is assumed that the reversible dimerization is a "head to tail" process (Case 1) or a "head to head" one (Case 2). The average values of K_s obtained for the GlcNAc-protein complex are: assuming Case 1 at 20°, (2.1 ± 0.7) x 10^-2 m and at 13°, (1.0 ± 0.4) x 10^-2 m; for Case 2 at 20°, (7.5 ± 2.8) x 10^-2 m and at 13°, (4.8 ± 1.7) x 10^-2 m. The saccharides GlcNAc-GlcNAc and GlcNAc-GlcNAc-GlcNAc are hydrolyzed slowly during sedimentation equilibrium so that equilibrium, presumably, was not reached. The values of K_s calculated for GlcNAc-GlcNAc and GlcNAc-GlcNAc-GlcNAc were approximately an order of magnitude larger than those obtained by dialysis. The values of K_s obtained when Case 1 is assumed for GlcNAc agree much better with the ones obtained from equilibrium dialysis. Previous studies indicated that a group with a pK_a of 6.2 is affected by the dimerization of lysozyme and that this group is probably a carboxylate. Since the present study indicated that the active site is involved in the dimerization of lysozyme and crystallographic studies indicate that glutamic acid residue 35 lies in the "cleft" of the active region and that it probably has a high pK_a, a reasonable conclusion is that the ionizable group with a pK_a of 6.2 which is involved in dimerization is glutamic acid residue 35.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				G. P. Gorbenko, V. M. Ioffe, and P. K. J. Kinnunen Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association Biophys. J., July 1, 2007; 93(1): 140 - 153. [Abstract] [Full Text] [PDF]

				E. Garcia-Hernandez, R. A. Zubillaga, E. A. Chavelas-Adame, E. Vazquez-Contreras, A. Rojo-Dominguez, and M. Costas Structural energetics of protein-carbohydrate interactions: Insights derived from the study of lysozyme binding to its natural saccharide inhibitors Protein Sci., January 1, 2003; 12(1): 135 - 142. [Abstract] [Full Text] [PDF]