Phillips N F, Wood H G
Biochemistry. 1986 Apr 8;25(7):1644-9. doi: 10.1021/bi00355a030.
The pyrophosphoryl form of pyruvate, phosphate dikinase was prepared by incubation with adenosine 5'-[gamma-32P]triphosphate and isolated by gel chromatography. Previously a phosphorylated moiety had been isolated from the enzyme and was shown to be bound through a phosphoramidate linkage to the 3' nitrogen of a histidine residue [Spronk, A. M., Yoshida, H., & Wood, H. G. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4415]. This histidine residue has been considered to be the pyrophosphoryl and phosphoryl carrier between the three subsites of this enzyme. Previous attempts to isolate the putative [32P]pyrophosphohistidine have been unsuccessful due to the lability of the [32P]pyrophosphoryl-enzyme. By stabilization of the [32P]pyrophosphoryl-enzyme with diazomethane, it has been possible to isolate a [32P]-pyrophosphohistidine from the hydrolysates. To our knowledge this work constitutes the first direct demonstration of a pyrophosphorylated histidyl residue in an enzyme.
丙酮酸、磷酸二激酶的焦磷酸化形式通过与腺苷5'-[γ-32P]三磷酸一起温育制备，并通过凝胶色谱法分离。此前，已从该酶中分离出一个磷酸化部分，并表明它通过磷酰胺键与组氨酸残基的3'氮相连[斯普龙克，A.M.，吉田，H.，&伍德，H.G.（1976年）《美国国家科学院院刊》73，4415]。这个组氨酸残基被认为是该酶三个亚位点之间的焦磷酸化和磷酸化载体。由于[32P]焦磷酸化酶的不稳定性，此前分离假定的[32P]焦磷酸组氨酸的尝试均未成功。通过用重氮甲烷稳定[32P]焦磷酸化酶，已能够从水解产物中分离出[32P]焦磷酸组氨酸。据我们所知，这项工作首次直接证明了酶中存在焦磷酸化的组氨酰残基。
