title: Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure.
abstract: 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 (delta G degrees = -1.3 kcal.mol-1). From the temperature dependence of delta G degrees, the following thermodynamic parameters were estimated: delta H degrees = 1.6 kcal.mol-1 and delta S 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 A for the main chain atoms and 1.19 +/- 0.23 A for all the non-hydrogen atoms of residues 5-40 and residues 48-67.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan 66506, USA.
authors.forename: M
authors.initials: M
authors.lastname: Cai
authors.forename: Y
authors.initials: Y
authors.lastname: Gong
authors.forename: O
authors.initials: O
authors.lastname: Prakash
authors.forename: R
authors.initials: R
authors.lastname: Krishnamoorthi
mesh_terms: D001665:Binding Sites; D015956:Factor XIIa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism*; D014675:Vegetables / Q000737:chemistry*
chemical_list: C081870:CMTI protein, Cucurbita maxima; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D015956:Factor XIIa