Phillips N F
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
Biochemistry. 1988 May 3;27(9):3314-20. doi: 10.1021/bi00409a029.
Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.
来自谢氏丙酸杆菌的丙酮酸磷酸双激酶受到5'-异硫氰酸荧光素（FITC）的强烈抑制。失活的时间进程是双相的，但准一级速率常数对抑制剂浓度的依赖性表明，在共价修饰之前，抑制剂与酶形成了可逆复合物。底物/产物核苷酸对MgATP和MgAMP可防止酶失活，而在没有Mg2+的情况下，两种核苷酸均无效。此前，通过使用AMP的2',3'-二醛（oAMP），已表明共生拟杆菌的酶的ATP/AMP亚位点上的一个必需赖氨酸起作用[埃文斯，C.T.，戈斯，N.H.，&伍德，H.G.（1980年）《生物化学》19，5809]。FITC的抑制作用与MgAMP具有竞争性，多重抑制分析图表明oAMP和FITC的结合是相互排斥的。这些观察结果表明，FITC和oAMP在核苷酸结合位点结合，并且可能与被oAMP修饰的同一个反应性赖氨酸结合。通过高效液相色谱进行肽图谱分析，发现FITC是从ATP/AMP亚位点分离肽的合适探针。
