Catalytic and ligand binding properties of bovine trypsinogen and its complex with the effector dipeptide Ile-Val. A comparative study.
Steady-state and pre-steady-state kinetic data for the trypsinogen catalyzed hydrolysis of a series of synthetic substrates (i.e. p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids) have been obtained as a function of pH (3.4-8). Moreover, the effect of ethylamine on the hydrolysis of a neutral substrate and benzamidine binding have been extensively studied. In order to obtain direct information on the transition of trypsinogen to a beta-trypsin-like structure, the role of the effector dipeptide Ile-Val on the catalytic and ligand binding properties of the zymogen has been investigated. Kinetic and thermodynamic data for beta-trypsin and alpha-chymotrypsin are also reported for the purpose of an homogeneous comparison of the various (pro)enzymes. Under all the experimental conditions, kinetic data for (pro)enzyme catalysis are consistent with the minimum three-step mechanism: (formula; see text) involving the acyl intermediate E X P. In the presence of Ile-Val dipeptide, trypsinogen assumes catalytic and ligand binding properties that are reminiscent of activated beta-trypsin. This is at variance with free trypsinogen, which shows a alpha-chymotrypsin-like behavior. The large differences in the results of kinetic and thermodynamic measurements for free trypsinogen, as compared to its binary adduct with Ile-Val, can be ascribed to the substantial differences in the two molecular species, which include the spatial orientation of Asp189.
D000818:Animals; D001550:Benzamidines / Q000378:metabolism; D002417:Cattle; D004151:Dipeptides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005021:Ethylamines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D013379:Substrate Specificity; D013816:Thermodynamics; D014362:Trypsinogen / Q000378:metabolism*
D001550:Benzamidines; D004151:Dipeptides; D005021:Ethylamines; C041115:isoleucylvaline; D014362:Trypsinogen; C032157:benzamidine