Crull G B, Goff H M
Department of Chemistry, University of Iowa, Iowa City 52242.
J Inorg Biochem. 1993 May 15;50(3):181-92. doi: 10.1016/0162-0134(93)80024-4.
The interaction of lactoperoxidase, LPO, with its substrate, thiocyanate, SCN-, has been investigated by 13C and 15N NMR relaxation measurements. When 0.1 M SCN-, enriched with either 13C or 15N, was titrated with native ferric lactoperoxidase a large change in the spin-lattice relaxation time of the respective nucleus was observed. In the presence of saturating amounts of CN-, a high affinity ligand for the heme iron, a similar but much smaller change in the relaxation time for SCN- was found. Studies of the rate of carbon relaxation as a function of temperature have shown that thiocyanate is in fast exchange between a site on the enzyme and bulk solution. When LPO in either the absence or presence of CN- was titrated with SCN- a linear increase in the relaxation time was observed. Dissociation constants (Kd values) have been determined from a least-squares analysis of these data. Apparent distances between the heme iron of lactoperoxidase and either the carbon or nitrogen atoms of bound thiocyanate ion have been determined through application of the Solomon-Bloembergen equation. These distances demonstrate that the observed association does not involve iron-thiocyanate coordination, suggesting the possibility of an anion binding site.
通过¹³C和¹⁵N NMR弛豫测量研究了乳过氧化物酶（LPO）与其底物硫氰酸盐（SCN⁻）之间的相互作用。用天然铁乳过氧化物酶滴定富含¹³C或¹⁵N的0.1 M SCN⁻时，观察到相应原子核的自旋晶格弛豫时间发生了很大变化。在存在饱和量的CN⁻（血红素铁的高亲和力配体）的情况下，发现SCN⁻的弛豫时间有类似但小得多的变化。对碳弛豫速率随温度变化的研究表明，硫氰酸盐在酶上的一个位点与本体溶液之间快速交换。用SCN⁻滴定不存在或存在CN⁻的LPO时，观察到弛豫时间呈线性增加。通过对这些数据进行最小二乘法分析确定了解离常数（Kd值）。通过应用所罗门 - 布洛姆伯根方程确定了乳过氧化物酶的血红素铁与结合硫氰酸根离子的碳原子或氮原子之间的表观距离。这些距离表明观察到的缔合不涉及铁 - 硫氰酸盐配位，这表明可能存在一个阴离子结合位点。
