Structural determinants of the half-life and cleavage site preference in the autolytic inactivation of chymotrypsin.

The molecular mechanism of the autolysis of rat alpha-chymotrypsin B was investigated. In addition to the two already known autolytic sites, Tyr146 and Asn147, a new site formed by Phe114 was identified. The former two sites and the latter one are located in the autolysis and the interdomain loops, respectively. By eliminating these sites by site-directed mutagenesis, their involvement in the autolysis and autolytic inactivation processes was studied. Mutants Phe114-->Ile and Tyr146-->His/Asn147-->Ser, that had the same enzymatic activity and molecular stability as the wild-type enzyme, displayed altered routes of autolytic degradation. The Phe114-->Ile mutant also exhibited a significantly slower autolytic inactivation (its half-life was 27-fold longer in the absence and sixfold longer in the presence of Ca2+ ions) that obeyed a first order kinetics instead of the second order displayed by wild-type chymotrypsin inactivation. The comparison of autolysis and autolytic inactivation data showed that: (a) the preferential cleavage of sites followed the order of Tyr146-Asn147 --> Phe114 --> other sites; (b) the cleavage rates at sites Phe114 and Tyr146-Asn147 were independent from each other; and (c) the hydrolysis of the Phe114-Ser115 bond was the rate determining step in autolytic inactivation. Thus, it is the cleavage of the interdomain loop and not of the autolysis or other loops that determines the half-life of chymotrypsin activity.

Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis.

Hereditary pancreatitis, an autosomal dominant disease is believed to be caused by mutation in the human trypsinogen gene. The role of mutations has been investigated by in vitro studies using recombinant rat and human trypsinogen (TG). In this study we compare the enzymatic properties and inhibition by human pancreatic secretory trypsin inhibitor (hPSTI) of the native, postsynthetically modified and recombinant cationic trypsin, and found these values practically identical. We also determined the autolytic stability of recombinant wild type (Hu1Asn21) and pancreatitis-associated (Hu1Ile21) trypsin. Both forms were equally stable. Similarly, we found no difference in the rate of activation of the two zymogens by human cationic and anionic trypsin. Mesotrypsin did not activate either form. The rate of autocatalytic activation of Hu1Asn21 TG and Hu1Ile21 TG was also identical at pH 8 both in the presence and absence of Ca2+. At pH 5 Hu1Ile21 TG autoactivated about twice as fast as Hu1Asn21 TG. The presence of physiological amount of hPSTI completely prevented autoactivation of both zymogens at pH 8 and at pH 5 as well. Cathepsin B readily activated both zymogens although Hu1Ile21 TG was activated about 2.5-3 times as fast as Hu1Asn21 TG. The presence of hPSTI did not prevent the activation of zymogens by cathepsin B. Our results underlie the central role of cathepsin B in the development of different forms of pancreatitis.

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms.

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).

The differential specificity of chymotrypsin A and B is determined by amino acid 226.

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydrophobic residues, like tryptophan, phenylalanine and tyrosine. We found, however, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phenylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all these substrates with comparable catalytic efficiency. Analysing the structure of substrate binding pocket of chymotrypsin A prompted us to perform an Ala226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsible for the differential specificities of chymotrypsin A and B isoenzymes.

Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site.

The binding of human alpha1-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pKa' of His57 in the active site but not to disrupt the hydrogen bond between His57 and Asp102. Similar NMR results were observed for the Asp189 to serine mutant of rat trypsin, which is much more stable than wild-type trypsin against autoproteolysis as the result of mutation of the residue at the base of the specificity pocket. This mutant was used in further studies aimed at determining the extent of the conformational transition in trypsin that accompanies serpin binding and leads to disruption of the catalytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat trypsin toward thermal denaturation was found to be lower in the free enzyme than in the complex with alpha1-proteinase inhibitor. This suggests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigations have shown that the proteinase in serpin-proteinase complexes becomes more susceptible to limited proteolysis, suggesting that the conformational change that accompanies binding leads to the exposure of susceptible loops in the enzyme. The existence of this type of conformational change upon complex formation has been confirmed here by investigation of the rate of cleavage of disulfide linkages by added dithiothreitol. This study revealed that, despite the increased stability of trypsin in the complex, one or more of its disulfide bridges becomes much more easily reduced. We suggest that the process of complex formation with alpha1-proteinase inhibitor converts trypsin D189S into an inactive, loose structure, which serves as a "conformational trap" of the enzyme that prevents catalytic deacylation. It is also proposed that plastic region(s) of the activation domain of trypsin may play a crucial role in this inhibitor-induced structural rearrangement.

Trypsin complexed with alpha 1-proteinase inhibitor has an increased structural flexibility.

Mutant rat trypsin Asp189Ser was prepared and complexed with highly purified human alpha 1-proteinase inhibitor. The complex formed was purified to homogeneity and studied by N-terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with alpha 1-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg117-Val118 one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.