CRYSTAL-STRUCTURE OF ESCHERICHIA-COLI PYRUVATE-KINASE TYPE-I - MOLECULAR-BASIS OF THE ALLOSTERIC TRANSITION
CRYSTAL-STRUCTURE OF ESCHERICHIA-COLI PYRUVATE-KINASE TYPE-I - MOLECULAR-BASIS OF THE ALLOSTERIC TRANSITION
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DOI：
10.1016/s0969-2126(01)00207-6
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发表时间：
1995-07-15
期刊：
影响因子：
5.7
通讯作者：
CODA, A
中科院分区：
文献类型：
--
作者：
MATTEVI, A;VALENTINI, G;CODA, A
背景：丙酮酸激酶(PK)在糖酵解的调节中起着重要作用。其催化活性由底物磷酸烯醇式丙酮酸和一个或多个变构效应物控制。猫和兔肌肉中非变构PKs的晶体结构是已知的。结果：未连接的I型PK在非活性T状态下的2.5埃分辨晶体结构表明，同源四聚体的每个亚基都包含一个(β/α)(8)-桶结构域、一个柔性的β-桶结构域和一个C-末端结构域。变构和活性中心位于结构域界面处。将T状态的大肠杆菌PK与被认为采用类似于活性R状态的构象的非变构肌酶进行比较，发现每个亚基的β-桶结构域和C-末端结构域的取向不同，分别旋转了17度和15度。此外，这四个亚基在两种酶中的相对取向相差约16度。亚基界面上高度保守的残基将这些运动与底物和变构效应结合部位的构象变化相结合。在T状态PK中观察到的亚基旋转导致(β/α)(8)桶结构域环6的移动，导致磷酸烯醇式丙酮酸结合部位的扭曲，这是T状态酶底物亲和力低的原因。结论：我们的结果表明PK的变构控制是通过显著的结构域和亚基旋转完成的。在从T态到R态的转变中，功能四聚体的所有12个结构域都改变了它们的相对取向。这些协同运动是活性中心和变构中心之间耦合的分子基础。
Background: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effecters. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia roll, in order to study the mechanism of allosteric regulation.Results: The 2.5 Angstrom resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)(8)-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)(8)-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme.Conclusions: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.