Dynamics of ligand binding to alpha-chymotrypsin and to N-methyl-alpha-chymotrypsin.
Ks values for binding of selected substrates, competitive inhibitors, and a noncompetitive inhibitor were found to be similar for alpha-chymotrypsin and N-methyl-alpha-chymotrypsin. The rates and steps of binding of a competitive inhibitor and a noncompetitive inhibitor were also found to be similar for alpha-chymotrypsin and N-methyl-alpha-chymotrypsin. Therefore, N-methyl-alpha-chymotrypsin is an appropriate model for alpha-chymotrypsin in studying the dynamics of the binding of substrates by temperature-jump techniques in aqueous solvents. 2-Toluidinylnaphthalene-6-sulfonate, a noncompetitive inhibitor, bound to alpha-chymotrypsin in a single step with rate constants k1 and k-1 of 3.9 X 10(7) M-1 s-1 and 1.9 X 10(3) s-1, respectively, at pH 5.0 (0.2 M acetate, ionic strength of 0.2). Similar values were obtained for N-methyl-alpha-chymotrypsin and chymotrypsinogen A at pH 5.0 and for alpha-chymotrypsin at pH 7.8 [0.1 M tris(hydroxymethyl)aminomethane-0.03 M CaCl2]. Indole, a competitive inhibitor, bound to alpha-chymotrypsin in a single step at pH 5.0 and 7.8, with k1 and k-1 of 1.8 X 10(7) M-1 s-1 and 7.8 X 10(3) s-1, respectively, at pH 5.0 while proflavin, another competitive inhibitor, bound to alpha-chymotrypsin with two observable steps where k1, k-1, k2, and k-2 were 1.0 X 10(7) M-1 s-1, 7 X 10(2) s-1, 1.0 X 10(3) s-1, and 7 X 10(2) s-1, respectively, at pH 5.0. The specific substrate N-acetyl-L-3,5-dinitrotyrosine ethyl ester bound to N-methyl-alpha-chymotrypsin at pH 5.0 in three observable steps where k1, k-1, k2, k-2, k3, and k-3 were 3.7 X 10(7) M-1 s-1, 6.2 X 10(4) s-1, 1.2 X 10(3) s-1, 3.5 X 10(2) s-1, 3 X 10(2) s-1, and 4 X 10(2) s-1, respectively. Preliminary data indicated that the third step of this reaction is probably absent when Met192 of N-methyl-alpha-chymotrypsin is oxidized to methionine sulfoxide. These results confirm the validity of data obtained from reactions at subzero temperatures in 65% dimethyl sulfoxide in indicating multiple steps in the binding of substrates to alpha-chymotrypsin. The methodology described should make it possible to measure quantitatively the contribution of the binding process to enzyme catalysis (the Circe effect).
D000818:Animals; D001667:Binding, Competitive; D002918:Chymotrypsin / Q000378:metabolism*; D002919:Chymotrypsinogen / Q000378:metabolism; D007700:Kinetics; D010059:Ovomucin; D011370:Proflavine; D013379:Substrate Specificity; D013696:Temperature; D014422:Turkeys
D010059:Ovomucin; D002919:Chymotrypsinogen; D011370:Proflavine; D002918:Chymotrypsin; C036178:N-methyl-alpha-chymotrypsin