Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy.
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DOI：
10.1021/bi000815k
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发表时间：
2000-08
期刊：
影响因子：
2.9
通讯作者：
D. Okar;D. Live;M. Devany;A. Lange
中科院分区：
文献类型：
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作者：
D. Okar;D. Live;M. Devany;A. Lange
用（15）N标记大鼠肝6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶的二磷酸酶结构域中的组氨酸，特异性地标记在N1'和全局上，用于异源单量子相关（HSQC）NMR光谱分析。通过与先前已指定的C2'质子的相关性来指定组氨酸相关的（15）N共振[Okar等人，Biochemistry 38，1999，4471-79]。从不含磷酸盐的样品中获得的（1）H-（15）N HSQC表明，以罕见的N1'互变异构体状态存在的His-258依赖于底物、果糖-2，6-二磷酸盐和随后的磷酸组氨酸中间体的O2磷酸盐填充的磷酸盐结合位点的占据。磷酸组氨酸中间体的特征在于涉及催化组氨酸His-258和His-392的两个氢键，其在咪唑环的N1'位置直接观察到。磷酸-His-258的N1'被质子化（（1）H化学位移，14.0 ppm），并与Gly-259的骨架羰基形成氢键。阳离子His-392的N1'与磷酸组氨酸的磷酰基部分形成氢键（（1）H化学位移，13.5 ppm）。质子化的磷酸-His-258中间体的存在和观察到与相同的磷酸组氨酸的相当强的氢键意味着共价中间体的水解在不需要任何“活化”水的情况下进行。使用标记的组氨酸作为Glu-327催化位点突变为丙氨酸的探针表明，除了在His-258的N3'处形成短暂的磷酸组氨酸中间体期间作为6-磷酸果糖的质子供体之外，该残基在维持催化位点的结构完整性方面具有重要作用。（1）H-（15）N HSQC数据还提供了明确的证据，尽管是表面残基，但His-446具有非常酸性的pK（a），远低于6.0。在这些观察的基础上，果糖-2，6-二磷酸酶的修正机制，这是与所有以前发表的动力学数据和X-射线晶体结构一致的建议。修订后的机制占催化组氨酸和Glu-327突变产生的结构和动力学后果。它还提供了一个假设的机制，通过cAMP依赖性磷酸化的Ser-32，这是位于N-末端激酶结构域的双磷酸酶激活的基础。
The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of these observations a revised mechanism for fructose-2,6-bisphosphatase that is consistent with all of the previously published kinetic data and X-ray crystal structures is proposed. The revised mechanism accounts for the structural and kinetic consequences produced by mutation of the catalytic histidines and Glu-327. It also provides the basis for a hypothetical mechanism of bisphosphatase activation by cAMP-dependent phosphorylation of Ser-32, which is located in the N-terminal kinase domain.