Núñez Sara, Wing Corin, Antoniou Dimitri, Schramm Vern L, Schwartz Steven D
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Phys Chem A. 2006 Jan 19;110(2):463-72. doi: 10.1021/jp051277u.
The catalytic site of the homotrimeric enzyme human purine nucleoside phosphorylase enzyme (hPNP) features residue F200 and the 241-265 loop directly skirting the purine base and a residue belonging to the adjacent monomer, F159, immediately conterminous to the ribosyl moiety. Crystallographic B-factors of apo human purine nucleoside phosphorylase, and hPNP complexed with substrate/transition state (TS) analogues, show that residue E250 is the centroid of a highly mobile loop region. Furthermore, superimposition of apo hPNP and hPNP complexed with TS analogue Immucillin-H shows a tightening of the active site, caused by the ligand-dependent 241-265 loop rearrangement taking place upon substrate/inhibitor binding, suggesting a putative dynamic role of the loop in binding/catalysis. However, crystallographic structures reveal only average atomic positions, and more detailed information is needed to discern the dynamic behavior of hPNP. The Essential Dynamics (ED) method is used here to investigate the existence of correlated motions in hPNP and consequently proposes mutagenesis assays to estimate the relative importance of these motions in the phosphorolytic efficiency of the reaction catalyzed by hPNP. We compare the concerted motions obtained from multiple molecular dynamics simulations of apo and Michaelis complex of hPNP both in vacuo and in solution. The results of the principal component analysis for the apo hPNP indicate the existence of strong correlations predominantly in the vicinity of residue F159. However, for the Michaelis complex, concerted motions are seen mostly around both active site residue F200 and loop residue E250. Additionally, for a simulation depicting the relaxation of tight complexed hPNP with a TS analogue, toward its relaxed apo form (after removal of the TS analog), a combination of the apo hPNP and Michaelis complex motions is found, with prominent concerted modes centered around neighboring residues F159, F200, and E250. Finally, we probed the extent to which these concerted motions bear an intrinsic catalytic role by performing experimental site-directed mutagenesis on some residues, followed by kinetic analysis. The F159G and F200G mutants displayed a strong increase in K(M) and modest decrease in k(cat), suggesting that these concerted motions may provide dynamical roles in substrate binding and/or catalysis. However, further structural data for the hPNP mutants are needed to confirm our hypothesis.
人嘌呤核苷磷酸化酶（hPNP）这一同三聚体酶的催化位点具有残基F200以及直接环绕嘌呤碱基的241 - 265环，还有来自相邻单体的一个残基F159，它紧邻核糖部分。无配体人嘌呤核苷磷酸化酶以及与底物/过渡态（TS）类似物复合的hPNP的晶体学B因子表明，残基E250是一个高度可移动环区域的质心。此外，无配体hPNP与与TS类似物Immucillin - H复合的hPNP的叠加显示，活性位点收紧，这是由底物/抑制剂结合时发生的依赖配体的241 - 265环重排引起的，表明该环在结合/催化中可能具有假定的动态作用。然而，晶体学结构仅揭示了平均原子位置，需要更详细的信息来辨别hPNP的动态行为。本文使用主成分分析（ED）方法来研究hPNP中相关运动的存在，并因此提出诱变分析以估计这些运动在hPNP催化反应的磷酸解效率中的相对重要性。我们比较了在真空和溶液中hPNP的无配体形式和米氏复合物的多个分子动力学模拟得到的协同运动。无配体hPNP的主成分分析结果表明，主要在残基F159附近存在强相关性。然而，对于米氏复合物，协同运动主要出现在活性位点残基F200和环残基E250周围。此外，对于一个描述与TS类似物紧密复合的hPNP向其松弛的无配体形式（去除TS类似物后）弛豫的模拟，发现了无配体hPNP和米氏复合物运动的组合，以相邻残基F159、F200和E250为中心有显著的协同模式。最后，我们通过对一些残基进行实验性定点诱变，然后进行动力学分析，探究了这些协同运动在多大程度上具有内在催化作用。F159G和F200G突变体的K(M)显著增加，k(cat)适度降低，表明这些协同运动可能在底物结合和/或催化中发挥动态作用。然而，需要hPNP突变体的进一步结构数据来证实我们的假设。
