Fluorescence monitoring of the conformational change in alpha(2)-macroglobulin induced by trypsin under second-order conditions: The macroglobulin acts both as a substrate and a competitive inhibitor of the protease

The reaction of bovine pancreatic trypsin with human plasma alpha(2)-macrog lobulin (alpha(2)M) was studied at 25 degrees C, using equimolar mixtures o f E and I in 50 mM potassium phosphate buffer, pH 7. The conformational cha nge in alpha(2)M was monitored through the increase in protein fluorescence at 320 nm (exc lambda, 280 nm). At [alpha(2)M](0) = [E](0) = 11.5-200 nM, the fluorescence change data fit the integrated second-order rate equation, (F-infinity - F-0)/(F-infinity - F-l) = 1 + k(i,obsd) [alpha(2)M](0)t, ind icating that cleavage of the bait region in alpha(2)M was the rate-determin ing step. The apparent rate constant (k(i.obsd)) was found to be inversely related to reactant concentration, The kinetic behavior of the system was compatible with a model involving reversible, nonbait region binding of E to alpha(2)M , competitively limiting the concentration of E available for bait region c leavage. The intrinsic value of k(i) was (1.7 +/- 0.24) x 10(7) M-1 s(-1). K-p, the inhibitory con stant associated with peripheral binding, was estim ated to be in the submicromolar range. The results of the present study point to a potential problem in interpreti ng kinetic data relating to protease-induced structural changes in macromol ecular substrates. If there is nonproductive binding, as in the case of try psin and alpha(2)M, and the reactions are monitored under pseudo first-orde r conditions ([S](0) much greater than [E](0)), an intrinsically second-ord er process (such as the rate-limiting bait region cleavage in alpha(2)M) ma y become kinetically indistinguishable from an intrinsically first-order pr ocess (e.g, rate-limiting conformational change). Hence an excess of one co mponent over the other should be avoided in kinetic studies addressing such systems.