Kasprzak A A, Villafranca J J
Department of Chemistry, Pennsylvania State University, University Park 16802.
Biochemistry. 1988 Oct 18;27(21):8050-6. doi: 10.1021/bi00421a012.
The interaction between Escherichia coli carbamoyl-phosphate synthetase (CPS) and a fluorescent analogue of an allosteric effector molecule, 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP), has been detected by using fluorescence techniques and kinetic measurements. From fluorescence anisotropy titrations, it was found that epsilon-AMP binds to a single site on CPS with Kd = 0.033 mM. The nucleotide had a small activating effect on the rate of synthesis of carbamoyl phosphate but had no effect on the Km for ATP. To test whether epsilon-AMP binds to an allosteric site, allosteric effectors (UMP, IMP, and CMP), known to bind at the UMP/IMP site, were added to solutions containing the epsilon-AMP-CPS complex. With addition of these effector molecules, a progressive decrease of the fluorescence anisotropy was observed, indicating that bound epsilon-AMP was displaced by the allosteric effectors examined. From these titrations, the dissociation constants for UMP, IMP, CMP, ribose 5-phosphate, 2-deoxyribose 5-phosphate, and orthophosphate were determined. When MgATP, a substrate, was employed as a titrant, the observed decrease in anisotropy was consistent with the formation of a ternary complex (epsilon-AMP-CPS-MgATP). The effect of ATP binding, monitored at the allosteric site, was magnesium dependent, and free magnesium in solution was required to obtain a hyperbolic binding isotherm. Solvent accessibility of epsilon-AMP in binary (epsilon-AMP-CPS) and ternary (epsilon-AMP-CPS-MgATP) complexes was determined from acrylamide quenching, showing that the base of epsilon-AMP is well shielded from the solvent even in the presence of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)
利用荧光技术和动力学测量方法，检测了大肠杆菌氨甲酰磷酸合成酶（CPS）与变构效应分子的荧光类似物1,N6-乙烯腺苷5'-单磷酸（ε-AMP）之间的相互作用。通过荧光各向异性滴定发现，ε-AMP以Kd = 0.033 mM的亲和力结合到CPS上的一个单一部位。该核苷酸对氨甲酰磷酸的合成速率有轻微的激活作用，但对ATP的Km值没有影响。为了测试ε-AMP是否结合到变构部位，将已知在UMP/IMP部位结合的变构效应物（UMP、IMP和CMP）添加到含有ε-AMP-CPS复合物的溶液中。随着这些效应分子的加入，观察到荧光各向异性逐渐降低，表明结合的ε-AMP被所检测的变构效应物取代。通过这些滴定，确定了UMP、IMP、CMP、5-磷酸核糖、2-脱氧核糖5-磷酸和正磷酸盐的解离常数。当使用底物MgATP作为滴定剂时，观察到的各向异性降低与三元复合物（ε-AMP-CPS-MgATP）的形成一致。在变构部位监测到的ATP结合效应依赖于镁，溶液中的游离镁是获得双曲线结合等温线所必需的。通过丙烯酰胺猝灭法测定了二元（ε-AMP-CPS）和三元（ε-AMP-CPS-MgATP）复合物中ε-AMP的溶剂可及性，结果表明即使在存在MgATP的情况下，ε-AMP的碱基也能很好地被溶剂屏蔽。（摘要截短于250字）
