Frick D N, Weber D J, Abeygunawardana C, Gittis A G, Bessman M J, Mildvan A S
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 1995 Apr 25;34(16):5577-86. doi: 10.1021/bi00016a032.
The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates to nucleoside monophosphates and pyrophosphate by substitution at the rarely attacked beta-phosphorus. Nucleotides containing bulky substituents at the 8 position of the purine ring are preferentially hydrolyzed. The conformation of the MutT-bound nonhydrolyzable substrate analog Mg(2+)-AMPCPP, determined by 10 intramolecular NOEs and molecular dynamics refinement using a full relaxation matrix analysis with back-calculation of the NOESY intensities, is high anti (chi = 53 +/- 9 degrees), with a C2'-exo, O1'-endo sugar pucker. Similarly, the product of dGTP hydrolysis, dGMP, also binds MutT in a high anti (chi = 73 +/- 9 degrees) C1'-endo conformation based on seven intramolecular NOEs. Such high anti rotations of the base would allow MutT to accommodate nucleotides substituted at the C-8 position with no intramolecular clashes. Changes in chemical shifts in the 1H-15N spectra of the enzyme induced by Mg2+ and Mg2+ AMPCPP suggest that the metal activator and nucleotide interact with residues in loop I, at the carboxyl end of helix I, loop II, loop III, and beta-strands A and B of the secondary structure of MutT. The displacement of Mg2+ by Mn2+ causes the selective disappearance due to paramagnetic broadening of 1H-15N cross peaks from G37, G38, and K39 in loop I and E57 in helix I. Eleven intermolecular NOEs between Mg2+AMPCPP and hydrophobic residues of MutT are found, three of which are tentatively assigned to L67 in loop II and three to L54 in helix I. Similarly, seven intermolecular NOEs between dGMP and hydrophobic residues of the enzyme are found, four of which are tentatively assigned to L54 and two to V58, both in helix I. These interactions indicate that the loop I-helix I-loop II motif contributes significantly to the active site of MutT in accord with mutagenesis studies and with sequence homologies among MutT-like NTP pyrophosphohydrolases.
MutT 酶通过取代很少被攻击的β-磷，催化核苷三磷酸水解为核苷单磷酸和焦磷酸。嘌呤环 8 位含有庞大取代基的核苷酸优先被水解。通过 10 个分子内 NOE 以及使用全弛豫矩阵分析并对 NOESY 强度进行反算的分子动力学精修确定的与 MutT 结合的不可水解底物类似物 Mg(2+)-AMPCPP 的构象为高反式（χ = 53 ± 9°），具有 C2'-外向、O1'-内向糖环构象。同样，dGTP 水解产物 dGMP 基于 7 个分子内 NOE 也以高反式（χ = 73 ± 9°）C1'-内向构象结合 MutT。碱基如此高的反式旋转将使 MutT 能够容纳在 C-8 位被取代的核苷酸而不会发生分子内冲突。Mg2+ 和 Mg2+ AMPCPP 诱导的酶的 1H-15N 谱化学位移变化表明，金属激活剂和核苷酸与 MutT 二级结构中 I 环、螺旋 I 羧基末端、II 环、III 环以及β链 A 和 B 中的残基相互作用。Mn2+ 取代 Mg2+ 导致 I 环中 G37、G38 和 K39 以及螺旋 I 中 E57 的 1H-15N 交叉峰因顺磁加宽而选择性消失。在 Mg2+AMPCPP 和 MutT 的疏水残基之间发现了 11 个分子间 NOE，其中 3 个暂定为 II 环中的 L67，3 个为螺旋 I 中的 L54。同样，在 dGMP 和酶的疏水残基之间发现了 7 个分子间 NOE，其中 4 个暂定为螺旋 I 中的 L54，2 个为 V58。这些相互作用表明，I 环 - 螺旋 I - II 环基序对 MutT 的活性位点有显著贡献，这与诱变研究以及 MutT 样 NTP 焦磷酸水解酶之间的序列同源性一致一致一致。
