Trypsinogen, Trypsin, Trypsin-Substrate and Trypsin-Inhibitor Complexes in Urea Solutions

The denaturation in urea of trypsinogen, trypsin and trypsin derivatives is first-order with respect to protein. 1 Structural modifications affecting the trypsinogen molecule were detected by studying the influence of pH on the rate of denaturation. The acidification of trypsinogen leads to the appearance of 2 reversible equilibria I′⇌ II′⇌ III′. I′ is the most stable form. However, form I of trypsin is more stable than form I′ of trypsinogen. The order of denaturation with respect to urea is 12 for I and 9 for I′. Forms III of trypsin and III′ of trypsinogen are denatured with the same rate and with the same order with respect to urea; this is additional evidence that in these forms the conformations of the zymogen and of the enzyme are very similar if not identical. 2 The catalytic mechanism of trypsin is the same in water or in urea solutions. Acylation and deacylation rates are unchanged below 7 M urea. Acylation (k2) is the rate-limiting step for amides; deacylation (k3) is limiting for esters (k2/k3=350). The free energy of binding of substrates and inhibitors to the specificity site is considerably decreased in urea solutions due to the diminished interaction between the hydrophobic parts of the substrate (or inhibitor) and of the specificity site. 3 The respective stabilities in urea of trypsin I, diisopropylphosphoro-trypsin I, trypsin-α-N-benzoly-l-argininé amide (Michaelis complex), trypsin-α-N-benzoyl-l-arginine ethyl ester (acyl enzyme), 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride-trypsin and trypsinbenzamidine have been compared. The stability of the enzyme increases with the number of binding points between the active site and the substrate (or inhibitor) and with the free energy of interaction between the apolar parts of the substrate (or inhibitor) and of the specificity site.