Properties of the His(57)-Asp(102) dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha(1)-proteinase inhibitor complexed forms

Structural and biochemical studies suggest that serpins induce structural r earrangements in their target serine-proteinases. Previous NMR studies of t he complex between a serpin, alpha(1)-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp(189) to Ser mutant, D189S, which is much more stable than wildtype rat trypsin against autoproteolysis) provided inf ormation about the state of catalytic residues in this complex: the hydroge n bond between Asp(102) and His(57) remains intact in the complex, and spec tral properties of His(57) are more like those of the zymogen than of the a ctivated enzyme (G;. Kaslik, et al., 1997, Biochemistry 36, 5455-5464), Her e we report the protonation and exchange behavior of His(57) Of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha(1)-proteinase inhibitor and compare these with an alogous behavior of His(57) of bovine chymotrypsinogen and alpha-chymotryps in. In these studies the pK(a) of His(57) has been determined from the pH d ependence of the H-1 NMR signal from the H-delta 1 proton of histidine in t he Asp(102)-His(57) dyad, and a measure of the accessibility of this part o f the active site has been obtained from the rate of appearance of this sig nal following its selective saturation. The activation of rat trypsinogen D 189S (zymogen, pK(a) = 7.8 +/- 0.1; Hill coefficient = 0.86 +/- 0.05) decre ased the pK(a) of His(57) by 1.1 unit and made the protonation process coop erative (active enzyme, pK(a) = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0. 08). The binding of alpha(1)-proteinase inhibitor to trypsin D189S led to a n increase in the pK(a) value of His(57) to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (comp lex, pK(a) = 8.1 +/- 0.1; Hill coefficient = 0.70 +/- 0.08), as was observe d for the zymogen. In spite of these differences in the pK(a) of His(57) in the zymogen, active enzyme, and alpha(1)-proteinase inhibitor complex, the solvent exchange lifetime of the His(57) H-delta 1 proton was the same, wi thin experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the H-1 NMR signal from the H-delta 1 proton of His(57) was r elatively 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, recombin ant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha(1)-proteinase inhibitor; however, in spectra of the complex between a lpha-chymotrypsin and human alpha(1)-proteinase inhibitor, the peak was bro ader and could be well-resolved only at the lower temperature (5 degrees C) . (C) 1999 Academic Press.