Alkaline structural transition of 4-nitrobenz-2-oxa-1,3-diazolyl-Lysozyme. Kinetic and spectroscopic investigations.
When lysozyme is reacted with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-CL), A 1:1 covalent product is produced, in which the NBD group arylates the phenolic hydroxyl group of Tyr-23 (Aboderin, A. A., and Boedefeld, E. (1976) Biochim. Biophys. Acta 420, 177). Changing the pH from neutral to alkaline conditions results in a large spectral shift of the absorption band associated with the NBD chromophore (Aboderin, A. A., Boedefeld, E., and Luisi, P. L. (1973) Biochim. Biophys. Acta 328, 30). In the present work it is shown that this spectral change is due to the formation of a sigma complex in which a hydroxyl ion is added to the aromatic nucleus of the nitrobenzoxadiazole system. Circular dichroic studies suggest that the NBD group is held in a conformationally rigid state in the protein. The kinetics of the spectral change accompanying the formation of the sigma complex has been investigated with a rapid mixing stopped flow spectrophotometer both in the modified enzyme and in the low molecular weight model compounds N-acetyl-(O-NBD)-L-tyrosinamide and glycyl-(O-NBD)-L-tyrosine. In the pH range from 10.1 to 12.7, the time course of the reaction is first order in the case of the modified enzyme (k = 4.8 s-1) and bimolecular and much slower (under pseudo-first order conditions) in the low molecular weight compounds. It is suggested that in the enzyme the reaction proceeds much faster because of the hydrophobic environment around the reacting groups. It is further suggested that the unimolecularity in the enzyme is due to a rate-determining isomerization step, probably connected with a local rearrangement of the protein conformation following the ionization of Tyr-20.
D009327:4-Chloro-7-nitrobenzofurazan; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D009113:Muramidase / Q000378:metabolism; D010069:Oxadiazoles; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D010069:Oxadiazoles; D009113:Muramidase; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine