Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.     

Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis.

Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca(2+) for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn(2+) EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn(2+) with a K(D) of 0.05 to 0.35 microM and one or more classes of lower affinity sites. Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five beta-sheet helices within the repeat domain of the toxin upon binding Ca(2+) is required for cell intoxication.     

Structural insights into the mechanism of nuclease A, a betabeta alpha metal nuclease from Anabaena

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the delta(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a betabeta alpha metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites approximately 26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the betabeta alpha metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.     

Structure of glutathione S-transferase of the filarial parasite Wuchereria bancrofti: a target for drug development against adult worm

A three dimensional structural model of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. The three dimensional X-ray crystal structure of porcine -class GST with PDB ID: 2gsr-A chain protein with 42% sequential and functional homology was used as the template. The model of wbGST built by MODELLER6v2 was analyzed by the PROCHECK programs. Ramachandran plot analysis showed that 93.5% of the residues are in the core region followed by 5.4 and 1.1% residues in the allowed and generously allowed regions, respectively. None of the non-glycine residues is in disallowed regions. The PROSA II z-score and the energy graph for the final model further confirmed the quality of the modeled structure. The computationally modeled three-dimensional (3D) structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668). 1SFM was used for docking with GST inhibitors by Hex4.2 macromolecular docking using spherical polar Fourier correlations.Figure: A three-dimensional (3D) structure of Glutathione-S-transferase (GST) of the lymphatic filarial parasite Wuchereria bancrofti (wb) was constructed by homology modeling. This modeled 3D structure of wbGST has been submitted to the Protein Data Bank (PDB) (PDB ID: 1SFM and RCSB ID: RCSB021668).     

Identification of potential inhibitors for HCV NS3 genotype 4a by combining protein–ligand interaction fingerprint, 3D pharmacophore,docking, and dynamic simulation

HCV NS3 protease domain has been one of the most attractive targets for developing new drugs for HCV infection and many drugs were successfully developed, but all of them were designed for targeting HCV genotype 1 infection. HCV genotype 4a dominant in Egypt has paid less attention. Here, we describe our protocol of virtual screening in identification of novel potential potent inhibitors for HCV NS3 of genotype 4a using homology modeling, PLIF (protein–ligand interaction fingerprint), docking, pharmacophore, and dynamic simulation. A high-quality 3D model of HCV NS3 protease of genotype 4a was constructed using crystal structure of HCV NS3 protease of genotype 1b (PDB ID: 4u01) as a template. PLIF was generated using five crystal structures of HCV NS3 (PDB ID: 4u01, 3kee, 4ktc, 4i33, and 5epn) which revealed the most important residues and their interactions with the co-crystalized ligands. A 3D pharmacophore model consisting of six features was developed from the generated PLIF data and then used as a screening filter for 11,244 compounds. Only 423 compounds passed the pharmacophore filter and entered the docking-based virtual screening stage. The highest ranked five hits from docking result (compound (C1–C5)) were selected for further analysis. They exhibited stronger interaction and higher binding affinity than HCV NS3 protease ligands. Dynamic simulation of the protein–best lead complex was performed to validate and augment the virtual screening results and it showed that these compounds have a strong binding affinity and could be very effective in treating HCV genotype 4a infections.     

Three-dimensional model of a substrate-bound SARS chymotrypsin-like cysteine proteinase predicted by multiple molecular dynamics simulations: catalytic efficiency regulated by substrate binding

Severe acute respiratory syndrome (SARS) is a contagious and deadly disease caused by a new coronavirus. The protein sequence of the chymotrypsin-like cysteine proteinase (CCP) responsible for SARS viral replication has been identified as a target for developing anti-SARS drugs. Here, I report the ATVRLQ(p1)A(p1')-bound CCP 3D model predicted by 420 different molecular dynamics simulations (2.0 ns for each simulation with a 1.0-fs time step). This theoretical model was released at the Protein Data Bank (PDB; code: 1P76) before the release of the first X-ray structure of CCP (PDB code: 1Q2W). In contrast to the catalytic dyad observed in X-ray structures of CCP and other coronavirus cysteine proteinases, a catalytic triad comprising Asp187, His41, and Cys145 is found in the theoretical model of the substrate-bound CCP. The simulations of the CCP complex suggest that substrate binding leads to the displacement of a water molecule entrapped by Asp187 and His41, thus converting the dyad to a more efficient catalytic triad. The CCP complex structure has an expanded active-site pocket that is useful for anti-SARS drug design. In addition, this work demonstrates that multiple molecular dynamics simulations are effective in correcting errors that result from low-sequence-identity homology modeling.     

In search of allosteric modulators of a7-nAChR by solvent density guided virtual screening

Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels whose activity can be modulated by endogenous neurotransmitters as well as by synthetic ligands that bind the same or distinct sites from the natural ligand. The subtype of α7 nAChR has been considered as a potenial therapeutic target for Alzheimer's disease, schizophrenia and other neurological and psychiatric disorders. Here we have developed a homology model of α7 nAChR based on two high resolution crystal structures with Brookhaven Protein Data Bank (PDB) codes 2QC1 and 2WN9 for threading on one monomer and then for building a pentamer, respectively. A number of small molecule binding sites are identified using Pocket Finder (J. An, M. Tortov, and R. Abagyan, Molecular & Cellular Proteomics, 4.6, 752-761 (2005)) of Internal Coordinate Mechanics (ICM). Remarkably, these computer-identified sites match perfectly with ordered solvent densities found in the high-resolution crystal structure of α1 nAChR, suggesting that the surface cavities in the α7 nAChR model are likely binding sites of small molecules. A high throughput virtual screening by flexible ligand docking of 5008 small molecule compounds was performed at three potential allosteric modulator (AM) binding sites of α7 nAChR using Molsoft ICM software (R. Abagyan, M. Tortov and D. Kuznetsov, J Comput Chem 15, 488-506, (1994)). Some experimentally verified allosteric modulators of α7 like CCMI comp-6, LY 7082101, 5-HI, TQS, PNU-120596, genistein, and NS-1738 ranked among top 100 compounds, while the rest of the compounds in the list could guide further search for new allosteric modulators.     

Homology modelling of a sensor histidine kinase from Aeromonas hydrophila

Aeromonas hydrophila has been implicated in extra-intestinal infection and diarrhoea in humans. Targetting unique effectors of bacterial pathogens is considered a powerful strategy for drug design against bacterial variations to drug resistance. The two-component bacterial system involving sensor histidine kinase (SHK) and its response regulators is considered a lucrative target for drug design. This is the first report describing a three-dimensional (3D) structure for SHK of A. hydrophila. The model was constructed by homology modelling using the X-ray structure of PleD—a response regulator—in conjunction with cdiGMP (PDB code 1W25) and HemAT sensor domain (PDB code 1OR4)—a globin coupled sensor. A combination of homology modelling methodology and molecular dynamics (MD) simulations was applied to obtain a reasonable structure to understand the dynamic behaviour of SHK. Homology modelling was performed using MODELLER9v2 software. The structure was relaxed to eliminate bad atomic contacts. The final model obtained by molecular mechanics and dynamics methods was assessed using PROCHECK and VERIFY 3D graph, which confirmed that the final refined model is reliable. Until complete biochemical and structural data of SHK are determined by experimental means, this model can serve as a valuable reference for characterising the protein and could be explored for drug targetting by design of suitable inhibitors.     

Homology model of the human tRNA splicing ligase RtcB

RtcB is an essential human tRNA ligase required for ligating the 2',3'‐cyclic phosphate and 5'‐hydroxyl termini of cleaved tRNA halves during tRNA splicing and XBP1 fragments during endoplasmic reticulum stress. Activation of XBP1 has been implicated in various human tumors including breast cancer. Here we present, for the first time, a homology model of human RtcB (hRtcB) in complex with manganese and covalently bound GMP built from the Pyrococcus horikoshii RtcB (bRtcB) crystal structure, PDB ID 4DWQA. The structure is analyzed in terms of stereochemical quality, folding reliability, secondary structure similarity with bRtcB, druggability of the active site binding pocket and its metal‐binding microenvironment. In comparison with bRtcB, loss of a manganese‐coordinating water and movement of Asn226 (Asn202 in 4DWQA) to form metal‐ligand coordination, demonstrates the uniqueness of the hRtcB model. Rotation of GMP leads to the formation of an additional metal‐ligand coordination (Mn‐O). Umbrella sampling simulations of Mn binding in wild type and the catalytically inactive C122A mutant reveal a clear reduction of Mn binding ability in the mutant, thus explaining the loss of activity therein. Our results furthermore clearly show that the GTP binding site of the enzyme is a well‐defined pocket that can be utilized as target site for in silico drug discovery.     

Improving the affinity and activity of CYP101D2 for hydrophobic substrates

CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate camphor to 5-exo-hydroxycamphor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and camphor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on camphor binding and the camphor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).     

The SuMo server: 3D search for protein functional sites

SUMMARY: We provide the scientific community with a web server which gives access to SuMo, a bioinformatic system for finding similarities in arbitrary 3D structures or substructures of proteins. SuMo is based on a unique representation of macromolecules using selected triplets of chemical groups having their own geometry and symmetry, regardless of the restrictive notions of main chain and lateral chains of amino acids. The heuristic for extracting similar sites was used to drive two major large-scale approaches. First, searching for ligand binding sites onto a query structure has been made possible by comparing the structure against each of the ligand binding sites found in the Protein Data Bank (PDB). Second, the reciprocal process, i.e. searching for a given 3D site of interest among the structures of the PDB is also possible and helps detect cross-reacting targets in drug design projects. AVAILABILITY: The web server is freely accessible to academia through http://sumo-pbil.ibcp.fr and full support is available from MEDIT (http://www.medit.fr). CONTACT: mjambon@burnham.org.     

Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase is a dimer of identical subunits. Previous work (Rapoport, T.A., et al. (1973) Eur. J. Biochem. 33, 341) indicated the presence of two different Mn2+ binding sites per subunit. In the present work, the binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex has been studied by 1H and 31P nuclear magnetic resonance. Two distinct phosphate sites have been found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 A) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analogue, hydroxymethanebisphosphonate, providing evidence for the physical proximity of these two sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analogue binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor.     

In silico model of DSF synthase RpfF protein from Xanthomonas oryzae pv. Oryzae: a novel target for bacterial blight of rice disease

BACKGROUND: Rice plant diseases play a major role as biological constraints on production. One of such rice disease is bacterial leaf blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo). The diffusible signal factor (DSF) synthesized by Xoo has a major role in virulence to rice plant. The DSF synthase RpfF protein, which is related to crotonase superfamily is responsible for the maintaining concentration of DSF. DSF-dependent quorum sensing (QS) system adopts protein- protein interaction mechanism to auto regulates the production of DSF. The antibacterial activity of pesticides against Xoo has not yet been completely understood. Three dimensional structure of RpfF protein was predicted using homology modeling method by MODELLER 9V9 software, SWISS MODEL and GENO3D online tools and structures were validated by Ramachandran plot, TM-Score and RMSD. 3D structure of RpfF (accession number AAL06345) was predicted using DSF synthase of Xanthomonas campestris pv. campestris (Xcc) (PDB ID: 3M6M) as a template. The stereo chemical check reveals the structure developed from the modeller was the best one and the potential ligand binding sites were identified by CASTp Server. The predicted RpfF model provides insight into its structure, active sites and aid in the development of novel inhibitors to control bacterial leaf blight in rice plant. DSF synthase RpfF protein could be used as a novel target to control infection.     

The manganese site of the photosynthetic oxygen-evolving complex probed by EPR spectroscopy of oriented photosystem II membranes: the g = 4 and g = 2 multiline signals.

The g = 4 and g = 2 multiline EPR signals arising from the Mn cluster of the photosynthetic oxygen-evolving complex (OEC) in the S2 state were studied in preparations of oriented photosystem II (PSII) membranes. The ammonia-modified forms of these two signals were also examined. The g = 4 signal obtained in oriented PSII membranes treated with NH4Cl at pH 7.5 displays at least 16 partially resolved Mn hyperfine transitions with a regular spacing of 36 G [Kim, D.H., Britt, R.D., Klein, M.P., & Sauer, K. (1990) J. Am. Chem. Soc. 112, 9389-9391]. The observation of this g = 4 "multiline signal" provides strong spectral evidence for a tetranuclear Mn origin for the g = 4 signal and is strongly suggestive of a model in which different spin state configurations of a single exchange-coupled Mn cluster give rise to the g = 4 and g = 2 multiline signals. A simulation shows the observed spectrum to be consistent with an S = 3/2 or S = 5/2 state of a tetranuclear Mn complex. The resolution of hyperfine structure on the NH3-modified g = 4 signal is strongly dependent on sample orientation, with no resolved hyperfine structure when the membrane normal is oriented perpendicular to the applied magnetic field. The dramatic NH3-induced changes in the g = 4 signal resolved in the spectra of oriented samples are suggestive that NH3 binding at the Cl- site of the OEC may represent direct coordination of NH3 to the Mn cluster.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploring CYP1A1 as anticancer target: homology modeling and in silico inhibitor design

Microsomal cytochrome P450 family 1 enzymes has great importance in the bioactivation of mutagens. P450 catalyzed reactions involve a wide range of substrates, and this versatality is reflected in a structural diversity, evident in the active sites of available P450 structures. This structure offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. In this paper, we document a homology model of CYP P450 1A1 from Homo sapiens, developed on the basis of template crystal structure of human microsomal P450 1a2 in complex with inhibitor (PDB Id: 2HI4). Homology modeling is performed at the programs, both in the commercial and public realms. We tried to explore CYP1A1 as a potential target for anticancer chemotherapy. To gain an insight into the binding of ligands with enzyme, protein-ligand complex was developed by including information about the known ligand as spatial restraints and optimizing the mutual interactions between the ligand and the binding site. Active site characterization and the study for involvement of specific aminoacids in binding with ligand, facilitates structure based inhibitor design. This study should prove useful in the design and development of potential novel anticancer agents. Figure The refined structure of homology model of CYP1A1     

An NMR and MD study of complexes of bacteriophage lambda lysozyme with tetra- and hexa-N-acetylchitohexaose

The X-ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa-N-acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E′F′ sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide. ¹H^N and ¹⁵N chemical shifts of λ lysozyme resonances were followed during NAG4/NAG6 titrations. The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme-inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme-peptidoglycan complex.     

Homology modeling of phosphoryl thymidine kinase of enterohemorrhagic Escherichia coli OH: 157

Enterohemorrhagic Escherichia coli (EHEC) are source of emerging infectious disease in India. Escherichia coli O157:H7 is an EHEC strain showing multiple antibiotic resistances and the cause of infantile diarrhea and hemolytic uremic syndrome worldwide. A novel strategy to counteract multiple antibiotic resistant organisms is to design drugs which specifically target metabolic pathways such as thiamine biosynthetic pathways found exclusively in prokaryotes. Homology modeling was used for model building of a terminal thiamine biosynthesis enzyme phosphoryl thymidine kinase (Thi E) using Geno3D, Swiss Model and Modeller. The best model was selected based on overall stereochemical quality. The potential ligand binding sites in the model were identified by CASTp server. The validated theoretical model of the 3D structure of the thiE protein of E. coli O157:H7 was predicted using a thiamine phosphate pyrophosphatase from Pyrococcus furiosus (PDB ID: 1X13_A) as template. The active pockets of ligand binding sites in the enzyme were identified. In this study, phosphoryl thymidine kinase (thi E), a terminal enzyme in the thiamine biosynthesis pathway in the pathogen has been modeled to be used in future as a potential drug target by the design of suitable inhibitors.     

Online homology modelling as a means of bridging the sequence-structure gap

For even the best-studied species, there is a large gap in their representation in the protein databank (PDB) compared to within sequence databases. Typically, less than 2% of sequences are represented in the PDB. This is partly due to the considerable experimental challenge and manual inputs required to solve three dimensional structures by methods such as X-ray diffraction and multi-dimensional nuclear magnetic resonance (NMR) spectroscopy in comparison to high-throughput sequencing. This gap is made even wider by the high level of redundancy within the PDB and under-representation of some protein categories such as membrane-associated proteins which comprise approximately 25% of proteins encoded in genomes. A traditional route to closing the sequence-structure gap is offered by homology modelling whereby the sequence of a target protein is modelled on a template represented in the PDB using in silico energy minimisation approaches. More recently, online homology servers have become available which automatically generate models from proffered sequences. However, many online servers give little indication of the structural plausibility of the generated model. In this paper, the online homology server Geno3D will be described. This server uses similar software to that used in modelling structures during structure determination and thus generates data allowing determination of the structural plausibility of models. For illustration, modelling of a chemotaxis protein (CheY) from Pseudomononas entomophila L48 (accession YP_609298) on a template (PDB id. 1mvo), the phosphorylation domain of an outer membrane protein PhoP from Bacillus subtilis, will be described.     

Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)