Dynamics of ligand binding to .alpha.-chymotrypsin and to N-methyl-.alpha.-chymotrypsin

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Abstract
Ks values for binding of selected substrates, competitive inhibitors and a noncompetitive inhibitor were 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 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 of 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 .times. 107 M-1 s-1 and 1.9 .times. 103 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)aminomethan.sbd.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 .times. 107 M-1 s-1 and 7.8 .times. 103 s-1, respectively, at pH 5.0 while proflavin, another competitive inhibitor, bound to .alpha.-chymotrypsin with 2 observable steps where k1, k-1, k2, and k-2 were 1.0 .times. 107 M-1 s-1, 7 .times. 102 s-1, 1.0 .times. 103 s-1, and 7 .times. 102 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 3 observable steps where k1, k-1, k2, k-2, k3, and k-3 were 3.7 .times. 107 M-1 s-1, 6.2 .times. 104 s-1, 1.2 .times. 103 s-1, 3.5 .times. 102 s-1, 3 .times. 102 s-1, and 4 .times. 102 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).