Regulation of alpha-chymotrypsin catalysis by ferric porphyrins and cyclodextrins

Positively charged alpha-chymotrypsin (ChT) formed a 1:1 complex with negatively charged 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) (FeTPPS) in phosphate buffer at pH 7.4 through electrostatic interaction. In spite of the large binding constant (K=4.8 x 10(5) M(-1)), FeTPPS could not completely inhibit the catalysis of ChT in the hydrolysis of the model substrate, N-succinyl-L-phenylalanine p-nitroanilide (SPNA). The degree of inhibition (60 %) was saturated at 1.6 equivalents of FeTPPS, which indicates that covering of the active site of ChT by FeTPPS was insufficient. The enzymatic activity lowered by FeTPPS was entirely recovered for the freshly prepared sample when the porphyrin on the protein surface was detached by per-O-methylated beta-cyclodextrin (TMe-beta-CD), which formed a stable 1:2 inclusion complex with FeTPPS (K(1)=1.26 x 10(6) M(-1), K(2)=6.3 x 10(4) M(-1)). FeTPPS gradually induced irreversible denaturation of ChT, and the denatured ChT further lost its catalytic ability. No repairing effect of TMe-beta-CD was observed with irreversibly denatured ChT. A new reversible inhibitor, 5,10,15,20-tetrakis[4-(3,5-dicarboxyphenylmethoxy)phenyl]porphyrinato iron(III) (FeP8M), was then designed, and its inhibitory behavior was examined. FeP8M formed very stable 1:1 and 1:2 FeP8M/ChT complexes with ChT, the K(1) and K(2) values being 2.0 x 10(8) and 1.0 x 10(6) M(-1), respectively. FeP8M effectively inhibited the ChT-catalyzed hydrolysis of SPNA (maximum degree of inhibition=85 %), and the activity of ChT was recovered by per-O-methylated gamma-cyclodextrin. No irreversible denaturation of ChT occurred upon binding with FeP8M. The kinetic data support the observation that, for nonincubated samples, both inhibitors did not cause significant conformational change in ChT and inhibited the ChT activity by covering the active site of the enzyme.