REACTIVE-SITE HYDROLYZED CUCURBITA-MAXIMA TRYPSIN INHIBITOR-V - FUNCTION, THERMODYNAMIC STABILITY, AND NMR SOLUTION STRUCTURE

Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima t rypsin inhibitor-V (CMTI-V) was prepared and characterized: In compar ison to the intact form, CMTI-V exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coag ulation factor XII(a). The equilibrium constant of trypsin-catalyzed h ydrolysis, K-hyd, defined as [CMTI-V]/[CMTI-V], was measured to be si milar to 9.4 at 25 degrees C (Delta G degrees = -1.3 kcal mol(-1)). Fr om the temperature dependence of Delta G degrees the following thermod ynamic parameters were estimated: Delta H degrees = 1.6 kcal . mol(-1) and Delta S degrees = 9.8 eu. In order to understand the functional a nd thermodynamic differences between the two forms, the three-dimensio nal solution structure of CMTI-V was determined by a combined approac h of NMR, distance geometry, and simulated annealing methods. Thus, fo llowing sequence-specific and stereospecific resonance assignments, in cluding those of beta-, gamma-, delta-, and epsilon-hydrogens and vali ne methyl hydrogens, 809 interhydrogen distances and 123 dihedral angl e constraints were determined, resulting in the computation and energy -minimization of 20 structures for CMTI-V. The average root mean squa red deviation in position for equivalent atoms between the 20 individu al structures and the mean structure obtained by averaging their coord inates is 0.67 +/- 0.15 Angstrom for the main chain atoms and 1.19 +/- 0.23 Angstrom for all the non-hydrogen atoms of residues 5-40 and res idues 48-67. Comparison of the mean structure of CMTI-V with the aver age NMR solution structure of CMTI-V [Cai, M., Gong, Y., Kao, J.-K.-F. , and Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211] indicated tertiary structural changes in the binding loop and N-terminal regions ; all the secondary structural elements were preserved. The newly form ed termini in CMTI-V are separated apart and more flexible. Structura l differences were reflected in the chemical shifts of the backbone hy drogen atoms and the pK(a) of His11 side chain, which changed from 5.5 8 +/- 0.02 in the intact form to 5.81 +/- 0.02 in the hydrolyzed form; the change in pK(a) is likely due to a stronger hydrogen bond, as ref lected by a shorter distance between (NH)-H-delta of His11 and main-ch ain oxygen of Pro10. The reduced binding affinities for trypsin and fa ctor XII(a) and increased entropy of CMTI-V are consistent with the i ncreased flexibility of the cleaved binding loop.