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Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, α1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189to 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 Asp102and His57remains intact in the complex, and spectral properties of His57are more like those of the zymogen than of the activated enzyme (G. Kaslik,et al.,1997,Biochemistry36, 5455–5464). Here we report the protonation and exchange behavior of His57of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human α1-proteinase inhibitor and compare these with analogous behavior of His57of bovine chymotrypsinogen and α-chymotrypsin. In these studies the pKaof His57has been determined from the pH dependence of the1H NMR signal from the Hδ1proton of histidine in the Asp102–His57dyad, 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 pKaof His57by 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 α1-proteinase inhibitor to trypsin D189S led to an increase in the pKavalue of His57to 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 pKaof His57in the zymogen, active enzyme, and α1-proteinase inhibitor complex, the solvent exchange lifetime of the His57Hδ1proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the1H NMR signal from the Hδ1proton of His57was relatively sharp, at temperatures between 5 and 20°C at both low pH (5.2) and high pH (10.0), in spectra of bovine α-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human α1-proteinase inhibitor; however, in spectra of the complex between α-chymotrypsin and human α1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5°C).

Mutant rat trypsin Asp 189Ser was prepared and complexed with highly purified human α1-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 α1-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg 117-Val 118one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.

Mutant rat trypsin Asp189Ser was prepared and complexed with highly purified human α1‐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 α1‐proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg117‐Val118one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase‐substrate and serine proteinase‐serpin reactions are discussed.