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Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure
- Cai, M., Gong, Y.X., Prakash, O., Krishnamoorthi, R.
- Biochemistry 1995 v.34 no.38 pp. 12087-12094
- Cucurbita maxima, active sites, binding capacity, blood coagulation, entropy, geometry, humans, hydrogen, hydrogen bonding, hydrolysis, nuclear magnetic resonance spectroscopy, oxygen, temperature, trypsin, valine
- Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucurbita maxima trypsin inhibitor-V (CMTI-V*) was prepared and characterized: In comparison to the intact form, CMTI-V* exhibited markedly reduced inhibitory properties and binding affinities toward trypsin and human blood coagulation factor XIIa. The equilibrium constant of trypsin-catalyzed hydrolysis, Khyd, defined as [CMTI-V*]/[CMTI-V], was measured to be approximately 9.4 at 25 degrees C (deltaG degrees = -1.3 kcal.mol-1). From the temperature dependence of deltaG degrees, the following thermodynamic parameters were estimated: deltaH degrees = 1.6 kcal.mol-1 and deltaS degrees = 9.8 eu. In order to understand the functional and thermodynamic differences between the two forms, the three-dimensional solution structure of CMTI-V* was determined by a combined approach of NMR, distance geometry, and simulated annealing methods. Thus, following sequence-specific and stereospecific resonance assignments, including those of beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens, 809 interhydrogen distances and 123 dihedral angle constraints were determined, resulting in the computation and energy-minimization of 20 structures for CMTI-V*. The average root mean squared deviation in position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.15 angstroms for the main chain atoms and 1.19 +/- 0.23 +/- for all the non-hydrogen atoms of residues 5-40 and residues 48-67. Comparison of the mean structure of CMTI-V* with the average 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 formed termini in CMTI-V* are separated apart and more flexible. Structural differences were reflected in the chemical shifts of the backbone hydrogen atoms and the pKa of His11 side chain, which changed from 5.58 +/- 0.02 in the intact form to 5.81 +/- 0.02 in the hydrolyzed form; the change in pKa is likely due to a stronger hydrogen bond, as reflected by a shorter distance between NdeltaH of His11 and main-chain oxygen of Pro10. The reduced binding affinities for trypsin and factor XIIa and increased entropy of CMTI-V* are consistent with the increased flexibility of the cleaved binding loop.