Modification of the carboxypeptidase A active site residue Glu-270 prevents interaction with a protein protease inhibitor from Ascaris.
The involvement of two active site residues of carboxypeptidase A in binding a protein inhibitor from Ascaris was studied. Glu-270 was modified with N-ethyl-5-phenylisoxazolium-3'-sulfonate and Tyr-248 was modified with tetranitromethane or diazotized arsanilic acid. Modification of Glu-270 abolished protein inhibitor binding and Glu-270 was protected from modification when the enzyme was bound to the protein inhibitor. In contrast, modification of Tyr-248 did not abolish protein inhibitor binding, nor did such binding protect Tyr-248 from modification. The absorption isosbestic point of arsanilazocarboxypeptidase A (Tyr-248 chemically modified) underwent a blue shift from 428 to 416 nm when the modified enzyme was bound to the protein inhibitor between pH 7.7 and 9.0. The 416 nm isosbestic point is characteristic of the loss of interaction between modified Tyr-248 and the active site zinc ion. These results with a protein inhibitor can be compared to substrate catalysis in which Tyr-248 moves away from the active site zinc ion of carboxypeptidase A when substrate binds. The close association of Glu-270 with Ascaris inhibitor interaction is consistent with other results which show that of the active site residues, only the modification of Glu-270 completely abolishes catalysis.
D000818:Animals; D001200:Ascaris; D001557:Benzenesulfonates; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors*; D043422:Carboxypeptidases A; D005971:Glutamates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007555:Isoxazoles; D010080:Oxazoles; D011480:Protease Inhibitors / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D013329:Structure-Activity Relationship; D013774:Tetranitromethane; D014443:Tyrosine / Q000378:metabolism
D001557:Benzenesulfonates; D005971:Glutamates; D007555:Isoxazoles; D010080:Oxazoles; D011480:Protease Inhibitors; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D014443:Tyrosine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D013774:Tetranitromethane