Solution Structure of the Quaternary MutT−M2+−AMPCPP−M2+ Complex and Mechanism of Its Pyrophosphohydrolase Action,

Abstract

The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked β-P, to yield NMP and pyrophosphate. It requires two divalent cations, forming an active E−M²⁺−NTP−M²⁺ complex. The solution structure of the free enzyme consists of a five-stranded mixed β-sheet connected by loop I−α-helix I−loop II, by two tight turns, and by loop III and terminated by loop IV−α-helix II [Abeygunawardana, C., et al. (1995) Biochemistry 34, 14997−15005]. Assignments of backbone ¹⁵N and NH resonances and side chain ¹⁵N and NH₂ resonances of the quaternary complex were made by ¹H−¹⁵N HSQC titrations of the free enzyme with MgCl₂ followed by equimolar AMPCPP/MgCl₂. H_α assignments were made by ¹H−¹⁵N 3D TOCSY HSQC, and ¹H−¹³C CT-HSQC spectra and backbone and side chain ¹H and ¹³C assignments were made by 3D HCCH TOCSY experiments. Ligands donated by the protein to the enzyme-bound divalent cation, identified by paramagnetic effects of Co²⁺ and Mn²⁺ on CO(C)H spectra, are the carboxylate groups of Glu-56, -57, and -98 and the amide carbonyl of Gly-38. The solution structure of the complex was computed with XPLOR using a total of 2168 NOE and 83 φ restraints for the protein, 11 intramolecular NOEs for bound Mg²⁺AMPCPP, 22 intermolecular NOEs between MutT and AMPCPP, and distances from the enzyme-bound Co²⁺ to the three phosphorus atoms of Co³⁺(NH₃)₄AMPCPP from paramagnetic effects of Co²⁺ on their T₁ values. The fold of the MutT enzyme in the complex is very similar to that of the free enzyme, with minor changes in the metal and substrate binding sites. The adenine ring binds in a hydrophobic cleft, interacting with Leu-4 and Ile-6 on β-strand A and with Ile-80 on β-strand D. The 6-NH₂ group of adenine approaches the side chain NH₂ of Asn-119. This unfavorable interaction is consistent with the stronger binding by MutT of guanine nucleotides, which have a 6-keto group. The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the γ-CH₂ of Lys-39 of loop I. The metal−triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation. One of two intervening water ligands is well positioned to attack P_β with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand. Lys-39 which is positioned to interact electrostatically with the α-phosphoryl group may facilitate the departure of the leaving NMP. On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies. It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P_β of NTP substrates.