Time-dependent changes in the intrinsic cotton effects of concanavalin A.

The near ultraviolet (UV) circular dichroism (CD) of concanavalin A (Con A) exhibits a notable time dependence following Ca2+ binding, whereas in the far W CD region, a region characteristic of protein secondary structure, the spectrum does not change with time. Discrete conformational forms are observed in the near W CD which differ both in their inherent spectral properties and in their spectral response to EDTA. The apoprotein (P) and the EDTA-sensitive forms of cobalt-Con A (CPS) and cobalt-calcium-Con A (CaCPS) exhibit unique extinctions (eP = 133 degrees/ mol, ECPS = 198 degrees/mol, &aCPS = 125 degrees/ mol) as does the fully metallized EDTA-resistant form (&aCPR = 34 degrees/mol). EDTA-quench methods establish that the CaCPS species slowly evolves to the CaCPR form. The energy of activation associated with the CaCPS + CaCPR conversion = 23 kcal/mol. The conversion occurs with a forward rate constant of 1.3 x 10m2 s-l at pH 5.3, 25°C. The kinetic formulations postulated by Brown et. al(Brown, R. D., III, Brewer, C. F., and Koenig, S. H. (1977) Biochemistry 16, 38833896) are thus substantiated both experimentally and analytically. The spectroscopic effects observed in the near W CD which are associated with (i) binding of metals at both sites S1 and S2, (ii) Ca’+-induced time dependence and (iii) binding of a-methyl-D-mannoside, are all modulated by a symmetrical, broad 277 nm transition. A hypothetical structural model, which envisions a sensitive tyrosine reporter, is presented to account for the spectroscopic observations.