Reaction heat variation with pH in formation of the trypsin-soybean inhibitor complex.

M T Barnhill,C G Trowbridge

doi:10.1016/s0021-9258(19)41210-6

Abstract

The heat of reaction between beta-trypsin and Kunitz soybean inhibitor (STI) hasbeen measured at 5 degrees and 25 degrees from pH 4 to 8.5. Corresponding measuremenportion of tRNA-Gly2-GGA/G molecules isolated from E. coli cells. The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNA-Gly2-GGA/G(nucleotide position 38). Asecondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNA-Gly2-suA36(HA), suggesting that the enzymes responsible for this modification recognize the anticodon sequencesof prospective tRNA substrates. The creation of a missense-suppressing tRNA, tRNA-Gly2-suA36(HA), by an alteration of the anticodon sequence of tRNA-Gly2-GGA/G is analogous to mechanisms whereby other suppressor tRNAs have arisen. The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions may be recognized by the glycyl-tRNA synthetase; the involvement of theanticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNA-Gly2-suA36(HA).

Highlights

The reaction heat-pH relationship is described by a model which postulates that the apparent reaction heat is controlled by a single ionizing group present in the free inhibitor and in the complexed state
We find at pH 4 that equilibrium among the intermediates is reached within 90 s for the reaction of P-trypsin with STI, and is reached within 180 s when STI* is the starting reactant
Our calorimetric study shows that the endothermic heat effect associated with the reaction between P-trypsin and soybean inhibitor is “controlled” by apparent pK values near those found by Markley [6] for one of the inhibitor histidine residues

Summary

Introduction

The reaction heat-pH relationship is described by a model which postulates that the apparent reaction heat is controlled by a single ionizing group present in the free inhibitor and in the complexed state. Over the pH range 4.5 to 5.5, where the trypsin-soybean reaction heat is most sensitive to pH, the reaction heat for trypsin-chicken ovomucoid is constant at 0 + 1 kcal. Suggests that the same abnormal histidine which affects the native-cleaved inhibitor equilibrium is responsible for the reaction heat-pH relationship for complex formation. Equilibrium from pH 4 to 8; the entropy change is negative throughout the range, and combines with the exothermic enthalpy change to produce a small favorable free energy change for this peptide bond cleavage

Methods

Results

Conclusion