PubMed Health⌕ Search

Biomedical subjects

D N Frick

Publications and source records attributed to D N Frick.

At least 19 recordsLinked to original sources
PubMed2006

Understanding helicases as a means of virus control.

Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genome replication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, severe acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and human papillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decrease viral replication in cell culture and animal models. This review discusses recent progress in understanding the structure and function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the design of better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary and functional similarities, and the key mechanistic features of each group are noted. All helicases share a common motor function fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acid chain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by preventing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a required cofactor.

Antiviral Agents↗
PubMed2001

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization.

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

Amino Acid Sequence↗
PubMed2001

DNA primases.

DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.

DNA Primers↗
PubMed2000

Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6.

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.

Amino Acid Sequence↗
PubMed1999

Interaction of bacteriophage T7 gene 4 primase with its template recognition site.

The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5'-GGGTC-3', 5'-TGGTC-3', 5'-GTGTC-3', or 5'-TTGTC-3'). Although it is not copied into the oligoribonucleotides, the cytosine at the 3'-position is essential for synthesis and template binding. Two nucleotides flanking the 3'-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5'-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 microM. The tight DNA binding (<1 microM) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.

Bacteriophage T7↗
PubMed1999

Interaction of ribonucleoside triphosphates with the gene 4 primase of bacteriophage T7.

The primase fragment of bacteriophage T7 gene 4 protein catalyzes the synthesis of oligoribonucleotides in the presence of ATP, CTP, Mg(2+) (or Mn(2+)), and DNA containing a primase recognition site. During chain initiation, ATP binds with a K(m) of 0.32 mM, and CTP binds with a K(m) of 0.85 mM. Synthesis of the dinucleotides proceeds at a rate of 3.8/s. The dinucleotide either dissociates or is extended to a tetranucleotide. The primase preferentially inserts ribonucleotides forming Watson-Crick base pairs with the DNA template >200-fold more rapidly than other ribo- or deoxynucleotides. 3'-dCTP binds the primase with a similar affinity as CTP and is incorporated as a chain terminator at a rate (1)/(100) that of CTP. ATP analogues alpha,beta-methylene ATP, beta,gamma-methylene ATP, and beta,gamma-imido ATP are incorporated by the primase fragment at the 5'-ends of the oligoribonucleotides but not at the 3'-ends. A model is presented in which the primase fragment utilizes two nucleotide-binding sites, one for the initiating ATP and one for the nucleoside triphosphate which elongates the primer on the 3'-end. The initiation site binds ATP or oligoribonucleotides, whereas the elongation site binds ATP or CTP as directed by the template.

Adenosine Triphosphate↗
PubMed1999

Studies on the ADP-ribose pyrophosphatase subfamily of the nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance.

Four Nudix hydrolase genes, ysa1 from Saccharomyces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite.

Adenosine Triphosphatases↗
PubMed1998

An N-terminal fragment of the gene 4 helicase/primase of bacteriophage T7 retains primase activity in the absence of helicase activity.

Primase and helicase activities of bacteriophage T7 are present in a single polypeptide coded by gene 4. Because the amino terminal region of the gene 4 protein contributes to primase activity, we constructed a truncated gene 4 encoding the N-terminal 271-aa residues. The truncated protein, purified from cells overexpressing the protein, is a dimer in solution; the full-length protein is a hexamer. Although the fragment is devoid of dTTPase and helicase activities, it catalyzes template-directed synthesis of di-, tri-, and tetranucleotides. The rates for tetraribonucleotide synthesis and for dinucleotide extension on a 20-nucleotide template are similar for the full-length and truncated proteins. However, the activity of the primase fragment is unaffected by dTTP whereas the primase activity of the full-length protein is stimulated >14-fold. The primase fragment is defective in the interaction with T7 DNA polymerase in that primer synthesis cannot be coupled to DNA synthesis.

Bacteriophage T7↗
PubMed1998

Orf186 represents a new member of the Nudix hydrolases, active on adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH.

orf186, a new member of the Nudix hydrolase family of genes, has been cloned and expressed, and the protein has been purified and identified as an enzyme highly specific for compounds of ADP. Its three major substrates are adenosine(5')triphospho(5')adenosine, ADP-ribose, and NADH, all implicated in a variety of cellular regulatory processes, supporting the notion that the function of the Nudix hydrolases is to monitor the concentrations of reactive nucleoside diphosphate derivatives and to help modulate their accumulation during cellular metabolism.

Adenosine Diphosphate Ribose↗
PubMed1997

Solution structure of the quaternary MutT-M2+-AMPCPP-M2+ complex and mechanism of its pyrophosphohydrolase action.

The MutT enzyme (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP) by substitution at the rarely attacked beta-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 beta-sheet connected by loop I-alpha-helix I-loop II, by two tight turns, and by loop III and terminated by loop IV-alpha-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(alpha) 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 phi 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 beta-strand A and with Ile-80 on beta-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 gamma-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(beta) 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 alpha-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(beta) of NTP substrates.

Adenosine Triphosphate↗
PubMed1996

Escherichia coli orf17 codes for a nucleoside triphosphate pyrophosphohydrolase member of the MutT family of proteins. Cloning, purification, and characterization of the enzyme.

The product of the Escherichia coli orf17 gene is a novel nucleoside triphosphate pyrophosphohydrolase with a preference for dATP over the other canonical (deoxy)nucleoside triphosphates, and it catalyzes the hydrolysis of dATP through a nucleophilic attack at the beta-phosphorus to produce dAMP and inorganic pyrophosphate. It has a pH optimum between 8.5 and 9.0, a divalent metal ion requirement with optimal activity at 5 mM Mg2+, a Km of 0.8 mM and a kcat of 5.2 s-1 at 37 degrees C for dATP. dAMP is a weak competitive inhibitor with a Ki of approximately 4 mM, while PPi is a much stronger inhibitor with an apparent Ki of approximately 20 microM. The enzyme contains the highly conserved signature sequence GXVEX2ETX6REVXEEX2I designating the MutT family of proteins. However, unlike the other nucleoside triphosphate pyrophosphohydrolases with this conserved sequence, the Orf17 protein does not complement the mutT- mutator phenotype, and thus must serve a different biological role in the cell.

Amino Acid Sequence↗
PubMed1996

The role of Glu 57 in the mechanism of the Escherichia coli MutT enzyme by mutagenesis and heteronuclear NMR.

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.

Amino Acid Sequence↗
PubMed1995

Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase.

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 beta-P, to yield NMP and pyrophosphate. Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I--loop II motif, by two tight turns, and by loop III, and terminated by loop IV--alpha-helix II [Abeygunawardana et al. (1993) Biochemistry 32, 13071-13080; Weber et al. (1993) Biochemistry 32, 13081-13087). Complete side-chain assignments of 1H and 13C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments. A total of 1461 interproton proximities (11 per residue), obtained by 3D 15N-resolved NOESY-HSQC and 3D 13C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT 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 beta-sheet sandwiched between the two alpha-helices, forming an alpha+beta 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, 5577-5586].(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

A novel GDP-mannose mannosyl hydrolase shares homology with the MutT family of enzymes.

The product of the Escherichia coli orf1.9, or yefc, gene (GenBank accession number L11721) has been expressed under the control of a T7 promoter, purified to apparent homogeneity, and identified as a novel enzyme that hydrolyzes GDP-mannose or GDP-glucose to GDP and the respective hexose. The enzyme has little or no activity on other nucleotides, dinucleotides, nucleotide sugars, or sugar phosphates. It has a pH optimum between 9.0 and 9.5, a Km of 0.3 mM, and a Vmax of 1.6 mumol min-1 mg-1 for GDP-mannose, and it requires divalent cations for activity. This enzyme of 160 amino acids (M(r) = 18, 405) contains the consensus sequence GX(I/L/V)(E/Q)(X)2ET(X)6R(X)4E(X)2(I/L), characteristic of the MutT family of proteins and previously shown to form part of the nucleotide-binding site of MutT (Frick, D. N., Weber, D. J., Abeygunawardana, C., Gittis, A. G., Bessman, M. J., and Mildvan, A. S. (1995) Biochemistry 34, 5577-5586). A comparison of the enzymatic reactions catalyzed by the GDP-mannose mannosyl hydrolase and the other enzymes of the MutT family suggests that the consensus signature sequence designates a novel nucleoside diphosphate binding site and catalytic motif.

Amino Acid Sequence↗
PubMed1995

NMR studies of the conformations and location of nucleotides bound to the Escherichia coli MutT enzyme.

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.

Adenosine Triphosphate↗
PubMed1995

Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes.

An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (M(r) = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 mumol min-1 mg-1 mM-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Km for NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5'-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 microM.

Amino Acid Sequence↗
PubMed1994

Dual divalent cation requirement of the MutT dGTPase. Kinetic and magnetic resonance studies of the metal and substrate complexes.

Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations. Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM). Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM). Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species. The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods. MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM. The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM. MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM. The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex. By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM. These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively. Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement.

Adenosine Triphosphate↗