JBC, Vol. 252, Issue 8, 2507-2514, Apr, 1977
Alkaline structural transition of 4-nitrobenz-2-oxa-1,3-diazolyl-Lysozyme. Kinetic and spectroscopic investigations
R. H. Sigg, P. L. Luisi and A. A. Aboderin
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.
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