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

Abstract

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).

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

“…The limit of k,,,/K, = kl kzlk-+ k 2 that might be approached in the tissue-kallikrein-catalyzed hydrolysis of the best ester substrates might well be k l , the rate of association of the substrate with the enzyme to form the Michaelis complex. For chymotrypsin, values of (1-24)x107 M -' s-' have been determined for k l [49,70,711, of the same order of magnitude asthevaluesof3.75or4.71 x107 M -' s -' of k,,,/K, obtained presently for the best dipeptide or tripeptide ester substrate of tissue kallikrein. k,,,/K, approaching k l requires k -to be smaller than or at most of similar magnitude to k2 [45].…”

Total synthesis of the polyether antibiotic ionomycin

“…Caled for C18H30O3Si: C, 67.03; H, 9.38. Found: C,67.29;H,9.56. (2S,3S)-3-((l,l-Dimethylethyl)dimethylsiloxy)-2-methyl-l-butanol (12). To a cooled (-78 °C), stirred solution of 18.50 g (57.36 mmol) of ester 11 in CH2C12 (100 mL) was added 126 mL (126 mmol) of a 1.0 M solution of diisobutylaluminum hydride over a 15-min period.…”

“…5% EtOAc/hexane) to afford a colorless oil, 1.64 g (79% from 12). Olefin isomer analysis before and/or after chromatography (DB-1, 150 °C, 10 psi, Z/2Z-13) = 2.87 min, ?,(13) = 3.79 min) revealed a ratio of 2Z-13:13 of 1.5:98.…”

“…(4/?,6S,8S,llS,12/?,14fl,16£,18l?,19! ?,20S,21S,22(2S,5S-(2/?,5S(£)))) 6,8,12,14,18,dioxy)- 9-oxo-22-(tetrahydro-5-methyl-5-(tetrahydro-5-(l-((l,l-dimethylethyl)dimethylsiloxy)ethyl)-5-methyl-2-furanyl)-2furanyl)-16-docosenoic Acid, Methyl Ester (57) and 11 £-57. Boron-Mediated Aldol Reaction.…”

“…In the interest of convergency, the decision was made to section this portion of the molecule at the C22-C23 bond, a plan that necessitates the creation of the C23 stereocenter in conjunction with subunit assemblage (transforms C and D). On the basis of a rationale that will be presented later, we projected that this stereocenter might be in- (12) Toeplitz, B. K.; Cohen, A. I.; Funke, P. T.; Parker, W. L; Gougoutas, synthon. Collectively, these transforms provided the four illustrated subunits of comparable complexity.…”