NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.

Weber, Darin J.; Bhatnagar, S. K.; Bullions, L C; Bessman, Maurice J.; Mildvan, Albert S.

doi:10.1016/s0021-9258(18)41875-3

Cited by 44 publications

(44 citation statements)

References 24 publications

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“…Kinetic Properties of the E57Q Mutant Compared with Wild Type MutT. No catalytic activity of the highly purified E57Q mutant of MutT was detected using three assays of progressively increasing sensitivity: a colorimetric assay (Bhatnagar et al, 1991), an NMR assay (Weber et al, 1992), and a radioactivity assay (Bhatnagar et al, 1991; see Materials and Methods). The most sensitive radioactivity assay revealed the E57Q mutant to be at least 10 5 -fold less active than the wild type enzyme over a range of pH values from 6.0 to 10.0.…”

Section: Resultsmentioning

confidence: 99%

“…The MutT enzyme from Escherichia coli, a pyrophosphohydrolase of 129 residues, catalyzes the unusual hydrolysis of nucleoside and deoxynucleoside triphosphates (NTP) by nucleophilic substitution at the rarely attacked β-phosphorus, yielding pyrophosphate and a nucleotide (NMP) as products (Bhatnagar et al, 1991;Weber et al, 1992). Like other enzymes which catalyze substitution at the electron-rich β-phosphorus, this enzyme requires two divalent cations for activity and forms a MutT-M 2+ -NTP-M 2+ complex (Frick et al, 1994).…”

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confidence: 99%

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The Role of Glu 57 in the Mechanism of the Escherichia coli MutT Enzyme by Mutagenesis and Heteronuclear NMR

et al. 1996

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The role of the conserved residue Glu-57 in the mechanism of the MutT enzyme from Escherichia coli was investigated by mutagenesis and heteronuclear NMR methods. The enzymatic activity of the E57Q mutant is at least 10(5)-fold lower than that of the wild type enzyme. The solution structure of the E57Q mutant, based on comparisons of 1H-15N NOESY HSQC spectra and 1H-15N HSQC spectra to those of the wild type enzyme, differs in a region near Glu-57. The dissociation constants (KD) of the E-Mg2+ and E-Mn2+ complexes increased 3.3- and 3.6-fold, respectively, in the E57Q mutant, while the KD of E-dGTP is unaltered from that of the wild type enzyme. The enhanced paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons is halved in the E57Q mutant indicating an altered metal-binding site. 1H-15N HSQC titrations of E57Q with MnCl2 show selective attenuation of the side chain NH signals of Gln-57 and the backbone NH signals of Gly-37, Gly-38, Lys-39, Glu-53, Glu-56, Gln-57, and Glu-98, indicating proximity of bound Nm2+ to these residues. The same resonances of the wild type and the E57Q mutant enzymes are attenuated by Mn2+, but significantly smaller paramagnetic effects (relative to the largest effect on Lys-39) are found on Gly-37, Gly-38, Val-58, and Glu-98 of the mutant, indicating an altered position of the bound divalent cation. Thus Glu-57 is probably a ligand to the enzyme-bound metal, and the profound loss of catalytic activity in the E57Q mutant results from structural and electronic changes at the site of the enzyme-bound divalent cation. 1H-15N HSQC titrations of the wild type enzyme with MgCl2 show changes in chemical shifts of 15N and NH resonances in regions closely overlapping those induced by the E57Q mutation itself, suggesting that the loss of the negative charge at Glu-57, either by mutation or by neutralization with Mg2+, induces a similar effect. In the E57Q mutant, the slow exchange of the side chain NH2 protons of Gln-57 and NOE's from the NH2 protons of Gln-57 to the beta and gamma protons of Glu-98 suggests hydrogen bonding of Gln-57 to Glu-98 in the free enzyme. 1H-15N HSQC titrations of both the wild type and mutant enzymes with dGTP show changes in 15N and NH chemicals shifts of residues in a cleft formed by beta-strands A, C, and D on one side and loop I, the end of loop IV, and the beginning of helix II on the other side, suggesting this cleft to be the nucleotide binding site. These changes in chemical shift were smaller or absent in titrations of the wild type or mutant enzymes with AMPCPP or Mg2+-AMPCPP, in accord with the strong preference of the MutT enzyme for guanine over adenine nucleotide substrates.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

The Role of Glu 57 in the Mechanism of the Escherichia coli MutT Enzyme by Mutagenesis and Heteronuclear NMR

et al. 1996

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“…The MutT enzyme, a pyrophosphohydrolase of 129 residues, catalyzes the unusual hydrolysis of nucleoside and deoxynucleoside triphosphates (NTP) by nucleophilic substitution at the rarely attacked /3-phosphorus, yielding pyrophosphate and a nucleotide (NMP) as products (Bhatnagar et al, 1991;Weber et al, 1992). Like other enzymes which catalyze substitution at the electron-rich /3-phosphorus, this enzyme requires two divalent cations for activity (Frick et al, 1994).…”

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confidence: 99%

Solution Structure of the MutT Enzyme, a Nucleoside Triphosphate Pyrophosphohydrolase

Abeygunawardana¹,

Weber²,

Gittis³

et al. 1995

Biochemistry

124

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The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked P-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed P-sheet connected by the loop I-a-helix I-loop I1 motif, by two tight tums, and by loop 111, and terminated by loop IV-a-helix I1 (4) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure ofMutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR. The structure is globular and compact with the parallel portion of the P-sheet sandwiched between the two a-helices, forming an a f , ! ? fold. The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods [Frick et al. (1995) Biochemistry 34, 5577-55861. The tertiary structure of MutT shows these residues to be near each other along the loop I-helix I region of the enzyme. A cluster of five glutamate residues (41,53, 56,57, and 98) form a patch of strongly negative electrostatic potential likely constituting the metal binding site. This site is contiguous with a deep cleft between P-strands A, C, and D and loop I which may contribute to the nucleotide binding site. This location of the active site is consistent with mutagenesis studies and with sequence homologies among MutT-like pyrophosphohydrolases.The MutT enzyme, a pyrophosphohydrolase of 129 residues, catalyzes the unusual hydrolysis of nucleoside and deoxynucleoside triphosphates (NTP) by nucleophilic substitution at the rarely attacked ,!?-phosphorus, yielding pyrophosphate and a nucleotide (NMP) as products (Bhatnagar et al., 1991;Weber et al., 1992). Like other enzymes which catalyze substitution at the electron-rich ,!?-phosphorus, this enzyme requires two divalent cations for activity (Frick et al., 1994). The biological role of this enzyme is to prevent ' This work was supported in part by National Institutes of Health Grants DK28616 (to A.S.M.), GM18649 (to M.J.B.), and GM36358 (which supports A.G.G.).A complete listing of the distance restraints derived from the NOE data has been deposited at the Brookhaven Protein Data Bank (Chemistry Department, Brookhaven National Laboratory, Upton, NJ) together with the atomic coordinates of the family of 15 acceptable structures (file name IMUT).

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“…Extensive mechanistic studies of E. coli MutT have shown the enzyme requires two magnesium ions for activity. , These divalent cations exhibit octahedral coordination involving conserved glutamate residues and a glycine from the conserved Nudix motif, the phosphate group of the substrate, and additional water molecules . Catalysis occurs by nucleophilic substitution by water at the internal β-phosphate atom, generating the mononucleotide form of the substrate and pyrophosphate . The reaction is accelerated by a glutamate residue from the Nudix motif, which acts as a general base to activate the attacking water molecule .…”

Section: Discussionmentioning

confidence: 99%

“…62 Catalysis occurs by nucleophilic substitution by water at the internal β-phosphate atom, generating the mononucleotide form of the substrate and pyrophosphate. 63 The reaction is accelerated by a glutamate residue from the Nudix motif, which acts as a general base to activate the attacking water molecule. 64 The existing structures of MutT in the Protein Data Bank with its preferred substrate 8-oxo-dG are all the monophosphate product (8-oxo-dGMP).…”

Section: ■ Discussionmentioning

confidence: 99%

Crystal Structure and Substrate Specificity of the 8-oxo-dGTP Hydrolase NUDT1 from Arabidopsis thaliana

et al. 2019

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NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction.

Cited by 44 publications

References 24 publications

The Role of Glu 57 in the Mechanism of the Escherichia coli MutT Enzyme by Mutagenesis and Heteronuclear NMR

The Role of Glu 57 in the Mechanism of the Escherichia coli MutT Enzyme by Mutagenesis and Heteronuclear NMR

Solution Structure of the MutT Enzyme, a Nucleoside Triphosphate Pyrophosphohydrolase

Crystal Structure and Substrate Specificity of the 8-oxo-dGTP Hydrolase NUDT1 from Arabidopsis thaliana

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