Disulfide bond-modified trypsinogen. Role of disulfide 179-203 on the specificity characteristics of bovine trypsin toward synthetic substrates.
Disulfide 179-203 of trypsinogen was cleaved and the free sulfhydryls were modified by S-carboxymethylation, S-carboxyamidomethylation, or by S-aminoethylation. The enzymatic properties of the activated, modified zymogens toward specific and nonspecific trypsin substrates were studies. The three S-alkylated trypsins hydrolyzed N-benzoylarginine ethyl ester and N-tosyl-lysine methyl ester with Kcat values similar to those of trypsin but with Km values that were increased by 2 to 3 orders of magnitude. The binding constant of the competitive inhibitor benzamidine to the S-alkylated trypsins was increased by 2 orders of magnitude by the modifications. The association constant of soybean trypsin inhibitor with S-carboxyamidomethyl trypsin was several orders of magnitude less than normal. Hydrolysis of benzoyl arginine amide with S-carboxymethyl trypsin was not detected, but this was ascribed to poor binding since a KI of 0.3 M was estimated from competitive inhibition studies. The altered kinetics did not depend on the type of chemical group used for the S-alkylated derivative, nor were the kinetics of any derivative significantly influenced by changes of pH or ionic strength. Nonspecific substrates, such as acetylglycine p-nitrophenyl ester and p-nitrophenylacetate, were hydrolyzed at equal rates by both trypsin and disulfide-modified trypsins, and both substrates had identical kcat/Km ratios with unmodified trypsins. For S-alkylated trypsins, kcat/km with lysine and arginine substrates were lower than normal and were the same order of magnitude as the values found for nonspecific substrates, suggesting normal catalytic behavior but a loss of specificity in binding substrates. The kinetic evidence suggested that the role of disulfide 179-203 in the mechanism of action of trypsin is to maintain the geometry of the specificity pocket by keeping appropriate residues of the pocket in a rigid framework.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004220:Disulfides / Q000032:analysis; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism*; D014362:Trypsinogen / Q000378:metabolism*
D004220:Disulfides; D014362:Trypsinogen; D014357:Trypsin