Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies.
Nucleotide regulation of Escherichia coli glycerol kinase: initial-velocity and substrate binding studies.
复制标题
大肠杆菌甘油激酶的核苷酸调节：初始速度和底物结合研究。
作者：
D. W. Pettigrew;G. Yu;Y. Liu
通过使用动力学和结合方法研究底物与大肠杆菌甘油激酶（EC 2.7.1.30；ATP-甘油3-磷酸转移酶）的结合。两个反应方向的初始速度研究显示了连续的动力学机制，ATP 明显激活底物，ADP 抑制底物。此外，米氏常数与底物解离常数有很大不同。使用 5'-腺苷酰亚胺二磷酸进行的产物抑制研究和死端抑制研究的结果表明，该酶具有随机动力学机制，这与观察到的与所有底物形成二元复合物以及该酶的不依赖于甘油的 MgATPase 活性一致。通过使用 N-乙基马来酰亚胺防止配体失活而确定的底物结合解离常数与初始速度研究中估计的解离常数一致。通过平衡透析测定底物结合化学计量显示一半位点结合 ATP、ADP 和甘油。因此，核苷酸的调节似乎并不反映在单独的调节位点处的结合。随机动力学机制消除了假设这样一个位点来解释与核苷酸形成二元复合物的需要。观察到的化学计量与核苷酸调节行为的模型一致，其中二聚体是测定中存在的酶形式，并且其亚基表现出不同的底物结合亲和力。酶的一些特性与负协同性一致，负协同性是亲和力差异的基础。考虑了 ATP 调节行为可能的生理重要性。
Substrate binding to Escherichia coli glycerol kinase (EC 2.7.1.30; ATP-glycerol 3-phosphotransferase) was investigated by using both kinetics and binding methods. Initial-velocity studies in both reaction directions show a sequential kinetic mechanism with apparent substrate activation by ATP and substrate inhibition by ADP. In addition, the Michaelis constants differ greatly from the substrate dissociation constants. Results of product inhibition studies and dead-end inhibition studies using 5'-adenylyl imidodiphosphate show the enzyme has a random kinetic mechanism, which is consistent with the observed formation of binary complexes with all the substrates and the glycerol-independent MgATPase activity of the enzyme. Dissociation constants for substrate binding determined by using ligand protection from inactivation by N-ethylmaleimide agree with those estimated from the initial-velocity studies. Determinations of substrate binding stoichiometry by equilibrium dialysis show half-of-the-sites binding for ATP, ADP, and glycerol. Thus, the regulation by nucleotides does not appear to reflect binding at a separate regulatory site. The random kinetic mechanism obviates the need to postulate such a site to explain the formation of binary complexes with the nucleotides. The observed stoichiometry is consistent with a model for the nucleotide regulatory behavior in which the dimer is the enzyme form present in the assay and its subunits display different substrate binding affinities. Several properties of the enzyme are consistent with negative cooperativity as the basis for the difference in affinities. The possible physiological importance of the regulatory behavior with respect to ATP is considered.
硫醇和氨基类似物作为来自念珠菌的甘油激酶的替代底物。
影响因子：
2.9
作者：
Knight,WB;Cleland,WW
通讯作者：
Cleland,WW
5-[p-(氟磺酰基)苯甲酰基]腺苷灭活大肠杆菌甘油激酶：水解试剂的保护。
影响因子：
2.9
作者：
Pettigrew,DW
通讯作者：
Pettigrew,DW