纤维素单胞菌嘌呤核苷磷酸化酶:由酶固有荧光的配体依赖性增强和配体对酶热失活的保护作用决定的理化性质和底物的结合

Beata Wielgus-Kutrowska , Agnieszka Bzowska , Jan Tebbe , Gertraud Koellner , David Shugar
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引用次数: 22

摘要

来自纤维素单胞菌sp.的嘌呤核苷磷酸化酶(PNP)在结晶状态下是三聚体,在溶液中也是三聚体。该酶的其他特征是典型的“低分子质量”PNPs,除了它在pH 11下不寻常的稳定性。嘌呤碱基、α-d-核糖-1-磷酸(R1P)和磷酸增强胞单胞菌PNP的固有荧光,从而形成二元配合物,诱导蛋白质构象改变,改变色氨酸残基的微环境。鸟嘌呤(Gua)结合的效果远远高于其他配体,表明该酶优先结合一种荧光的,很可能是罕见的Gua阴离子形式的互变异构,PNP/Gua配合物与Gua阴离子及其n -甲基衍生物的发射特性的比较进一步证明了这一点。鸟苷(Guo)和肌苷(Ino)在100 μM浓度下对酶的固有荧光影响较小,对酶的热失活没有影响,但它们对酶的保护作用表明它们与PNP形成弱的二元配合物。Gua、次黄嘌呤(Hx)和R1P与三聚体酶的结合用一个解离常数来描述,Gua、Hx和R1P的Kd值分别为0.46 μM、3.0 μM和60 μM。Gua(可能还有Hx)的结合化学计量是每个酶三聚体有三个配体分子。磷酸盐对酶固有荧光的影响不仅是由于结合,而且由于离子强度的增加,如用KCl滴定所示。在校正了离子强度的影响后,磷酸盐滴定数据与一个解离常数Kd=270 μM最一致,但不能明确排除Kd>50 mM的非常弱的结合位点的存在。瓜氨酸与PNP/磷酸二元配合物的结合(Kd=1.7 μM)弱于与游离酶的结合(Kd=0.46 μM),表明磷酸在催化解磷过程中有助于释放嘌呤碱。结果表明,PNP(包括胞单胞菌PNP)典型的初始速度非线性动力学图并不像通常认为的那样是由于单体之间的合作相互作用形成的,而是一个比迄今为止所考虑的更复杂的动力学机制。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence, and by protective effects of ligands on thermal inactivation of the enzyme

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for “low molecular mass” PNPs, except for its unusual stability at pH 11. Purine bases, α-d-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 μM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, Kd=0.46 μM for Gua, 3.0 μM for Hx, and 60 μM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, Kd=270 μM, but existence of a very weak binding site with Kd>50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker (Kd=1.7 μM) than to the free enzyme (Kd=0.46 μM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis.

The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.

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