Cámara-Artigas A, Parody-Morreale A, Barón C
Departamento de Química Física, Bioquímica y Química Inorgánica, Facultad de Ciencias Experimentales, Universidad de Almería, Spain.
Int J Biochem Cell Biol. 1997 May;29(5):849-56. doi: 10.1016/s1357-2725(96)00149-5.
The mechanism of activation of glycogen phosphorylase is incompletely understood, although adenosine and inosine nucleotides are known to be important allosteric activators. In this study the activation of glycogen phosphorylases a and b from bovine liver by adenosine 5'-monophosphate (AMP) and inosine 5'-monophosphate (IMP) has been investigated and the results compared with the activation of the muscle isozyme by the same nucleotides. Enzyme activity was determined by spectrophotometric measurement of inorganic phosphate produced in the phosphorylase-catalysed reaction of glycogen synthesis. Liver phosphorylase b binds both nucleotides non-co-operatively (Hill coefficients of 1.0 +/- 0.1), with changes in the maximum velocity to 75 or 80 mumol min-1 mg-1 in the presence of adenosine 5'-monophosphate or inosine 5'-monophosphate, respectively, but no change in the enzyme affinity towards the substrate, glucose-1-phosphate. Binding of glucose-1-phosphate is co-operative and the kinetic data have been fitted with the Monod-Wyman-Changeux model. Liver phosphorylase a has a maximum velocity similar to that of the b form in the presence of nucleotides. Binding of glucose-1-phosphate to the enzyme is non-co-operative (Hill coefficient of 1.0 +/- 0.1) and the affinities in the presence of the nucleotides (Michaelis constants of 28 +/- 0.2 mM or 27 +/- 0.2 mM for adenosine 5'-monophosphate or inosine 5'-monophosphate) are stronger than those of the b form. It is concluded that the activity of bovine liver phosphorylase a and b is similarly influenced by adenosine 5'-monophosphate or inosine 5'-monophosphate. The b form seems to behave like muscle phosphorylase b in response to inosine 5'-phosphate; however, the binding of adenosine 5'-phosphate does not induce the conformational change necessary to activate the liver enzyme, as occurs with the muscle isozyme.
尽管已知腺苷和肌苷核苷酸是重要的变构激活剂，但糖原磷酸化酶的激活机制仍未完全明确。在本研究中，对5'-单磷酸腺苷（AMP）和5'-单磷酸肌苷（IMP）激活牛肝糖原磷酸化酶a和b的过程进行了研究，并将结果与相同核苷酸对肌肉同工酶的激活情况进行了比较。通过分光光度法测定磷酸化酶催化糖原合成反应中产生的无机磷酸盐来确定酶活性。肝磷酸化酶b与两种核苷酸均为非协同结合（希尔系数为1.0±0.1），在存在5'-单磷酸腺苷或5'-单磷酸肌苷时，最大反应速度分别变为75或80μmol·min⁻¹·mg⁻¹，但酶对底物1-磷酸葡萄糖的亲和力未发生变化。1-磷酸葡萄糖的结合具有协同性，动力学数据已用莫诺德-怀曼-尚热模型进行拟合。在存在核苷酸的情况下，肝磷酸化酶a的最大反应速度与b型相似。1-磷酸葡萄糖与该酶的结合为非协同性（希尔系数为1.0±0.1），在存在核苷酸时的亲和力（5'-单磷酸腺苷或5'-单磷酸肌苷的米氏常数分别为28±0.2 mM或27±0.2 mM）比b型更强。得出的结论是，5'-单磷酸腺苷或5'-单磷酸肌苷对牛肝磷酸化酶a和b活性的影响相似。b型在对5'-磷酸肌苷的反应中似乎表现得与肌肉磷酸化酶b类似；然而，与肌肉同工酶不同，5'-磷酸腺苷的结合并未诱导激活肝酶所需的构象变化。
