Kinetic mechanism and pH dependence of the kinetic parameters of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase
Kinetic mechanism and pH dependence of the kinetic parameters of Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase
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DOI：
10.1006/abbi.2001.2618
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发表时间：
2001-12-01
影响因子：
3.9
通讯作者：
Tipton, PA
中科院分区：
文献类型：
--
作者：
Naught, LE;Tipton, PA
在人类致病菌中，磷酸甘露糖变位酶/磷酸葡萄糖变位酶(PMM/PGM)负责形成甘露糖1-磷酸和葡萄糖1-磷酸，甘露糖1-磷酸和葡萄糖I-磷酸是导致铜绿假单胞菌致病的多糖生物合成所必需的，因此PMM/PGM的抑制剂可能导致临床有用的化合物。甘露糖6-磷酸和葡萄糖6-磷酸的V/K值表明它们同样是该酶的良好底物。在大肠杆菌中过度表达的PMM/PGM是作为一种磷酸酶分离出来的；令人惊讶的是，丝氨酸108发生磷酸化的突变导致不同残基的磷酸化，从而使活性仅比野生型酶降低20倍。在反向反应中，葡萄糖1-磷酸表现出底物抑制，这是由于它与活化剂葡萄糖1，6-二磷酸竞争结合脱磷酸酶而产生的。这一现象与酶将底物磷酸化以生成双磷酸化中间体的机制一致，该中间体在活性部位重新定向，从而将其原始的磷酰基返回到酶中并生成观察到的产物。动力学参数的pH依赖关系表明，活性中心含有一个在催化反应中充当一般碱的残基和一个充当一般酸的残基。但是，总酸的pK为7.4，总碱的pK为8.4，因此这些残基在活性酶中以反质子化的状态存在。(C)2001年爱思唯尔科学公司。
The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) is responsible for the formation of mannose 1-phosphate and glucose 1-phosphate in the human pathogenic bacterium Pseudomonas aeruginosa Mannose 1-phosphate and glucose I-phosphate are required for the biosynthesis of polysaccharides that contribute to the virulence of P. aeruiginosa, so inhibitors of PMM/PGM may lead to clinically useful compounds. The V/K values for mannose 6-phosphate and glucose 6-phosphate show that they are equally good substrates for the enzyme. PMM/PGM overexpressed in Escherichia coli is isolated as a phosphoenzyme; surprisingly, mutation of serine 108 where phosphorylation occurs results in phosphorylation of a different residue so that activity is reduced only 20-fold from that of wild-type enzyme. In the reverse reaction glucose 1-phosphate exhibits substrate inhibition, which arises through its competition with the activator glucose 1,6-bisphosphate for binding to dephosphoenzyme. This phenomenon is consistent with a mechanism in which the enzyme phosphorylates the substrate to generate a bisphosphorylated intermediate that reorients in the active site to return its original phosphoryl group to the enzyme and generate the observed product. The pH dependence of the kinetic parameters suggests that the active site contains a residue that serves as a general base in the catalytic reaction and one that acts as a general acid. However, the pK of the general acid is 7.4 and that of the general base is 8.4 so these residues exist in a state of reverse protonation in the active enzyme. (C) 2001 Elsevier Science.