Modi S, Behere D V, Mitra S
Chemical Physics Group, Tata Institute of Fundamental Research, Colaba, Bombay, India.
Biochim Biophys Acta. 1989 Jul 6;996(3):214-25. doi: 10.1016/0167-4838(89)90250-1.
The interaction of aromatic donor molecules with lactoperoxidase (LPO) was studied using 1H-NMR and optical difference spectroscopy techniques. pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with pKa of 6.1) which is presumably a distal histidine. Dissociation constants evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements were found to be an order of magnitude larger than those for binding to horse radish peroxidase (HRP), indicating relatively weak binding of the donors to LPO. The dissociation constants evaluated in presence of excess of I- and SCN- showed a considerable increase in their values, indicating that the iodide and thiocyanate ions compete for binding at the same site. The dissociation constant of the substrate binding was, however, not affected by cyanide binding to the ferric centre of LPO. All these results indicate that the organic substrates bind to LPO away from the ferric center. Comparison of the dissociation constants between the different substrates suggested that hydrogen bonding of the donors with the distal histidine amino acid, and hydrophobic interaction between the donors and the active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the LPO-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative and relatively low compared to those for binding to HRP. The distances of the substrate protons from the ferric center were found to be in the range 9.4-11.1 A which are 2-3 A larger than those reported for the HRP-substrate complexes. These structural informations suggest that the heme in LPO may be more deeply buried in the heme crevice than that in the HRP.
利用¹H-NMR和光学差示光谱技术研究了芳香供体分子与乳过氧化物酶（LPO）的相互作用。底物质子共振线宽的pH依赖性表明，一个氨基酸残基（pKa为6.1，推测为远端组氨酸）的质子化促进了结合。从光学差示光谱和¹H-NMR弛豫测量评估得到的解离常数比与辣根过氧化物酶（HRP）结合的解离常数大一个数量级，表明供体与LPO的结合相对较弱。在过量I⁻和SCN⁻存在下评估的解离常数显示其值有相当大的增加，表明碘离子和硫氰酸根离子在同一位点竞争结合。然而，底物结合的解离常数不受氰化物与LPO铁中心结合的影响。所有这些结果表明有机底物在远离铁中心的位置与LPO结合。不同底物之间解离常数的比较表明，供体与远端组氨酸氨基酸的氢键作用以及供体与活性位点之间的疏水相互作用对缔合作用力有显著贡献。评估了与LPO-底物平衡相关的自由能、熵和焓的变化。发现这些热力学参数均为负值，且与与HRP结合的参数相比相对较低。发现底物质子与铁中心的距离在9.4 - 11.1 Å范围内，比报道的HRP-底物复合物的距离大2 - 3 Å。这些结构信息表明，LPO中的血红素可能比HRP中的血红素更深地埋在血红素裂隙中。
