Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure.
Reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor-V: function, thermodynamic stability, and NMR solution structure.
复制标题
反应位点水解南瓜胰蛋白酶抑制剂-V：功能、热力学稳定性和 NMR 溶液结构。
DOI：
10.1021/bi00038a001
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Krishnamoorthi,R
中科院分区：
文献类型：
--
作者：
Cai,M;Gong,Y;Prakash,O;Krishnamoorthi,R
Reactive-site (Lys44-Asp45 peptide bond) hydrolyzed Cucúrbita 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, Xhyd, defined as [CMTI-V*]/[CMTI-V], was measured to be~ 9.4 at 25 C (G=—1.3 kcabmol-1). From the temperature dependence of G, the following thermodynamic parameters were estimated: AH= 1.6 kcalmol-1 and AS=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 ß-,-,-, and e-hydrogens and valine methyl hydrogens, 809 interhydrogen distancesand 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 Á for the main chainatoms and 1.19±0.23 Á for all the non-hydrogen atoms of residues 5—40 and residues 48—67. Comparison of the mean structure of CMTIV* 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 His 11 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 N6H of His 11 and main-chain oxygen of Pro 10. The reduced bindingaffinities for trypsin and factor XIIa and increased entropy of CMTI-V*
