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)-macroglobulin (alpha(2)M) was studied at 25 degrees C, using equimolar mixtures of E and I in 50 mM potassium phosphate buffer, pH 7. The conformational change 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(t) )=1+k(i,obsd) [alpha(2)M](0) t, indicating that cleavage of the bait region in alpha(2)M was the rate-determining 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 cleavage. The intrinsic value of k(i) was (1.7+/-0.24) x 10(7) M(-1) s(-1).K(p), the inhibitory constant associated with peripheral binding, was estimated to be in the submicromolar range. The results of the present study point to a potential problem in interpreting kinetic data relating to protease-induced structural changes in macromolecular substrates. If there is nonproductive binding, as in the case of trypsin and alpha(2)M, and the reactions are monitored under pseudo first-order conditions ([S](0) >>[E](0) ), an intrinsically second-order process (such as the rate-limiting bait region cleavage in alpha(2)M) may become kinetically indistinguishable from an intrinsically first-order process (e.g. rate-limiting conformational change). Hence an excess of one component over the other should be avoided in kinetic studies addressing such systems.
Ozer
I
I
Department of Biochemistry, School of Pharmacy, Hacettepe University, 06100 Ankara, Turkey.
SimSek
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H
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002417:Cattle; D010450:Endopeptidases / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry*; D000511:alpha-Macroglobulins / Q000737:chemistry*
D000511:alpha-Macroglobulins; D010450:Endopeptidases; D014357:Trypsin