EcoRV restriction endonuclease: communication between catalytic metal ions and DNA recognition.

In the absence of magnesium ions, the EcoRV restriction endonuclease binds all DNA sequences with equal affinity but cannot cleave DNA. In the presence of Mg2+, the EcoRV endonuclease cleaves DNA at one particular sequence, GATATC, at least a million times more readily than any other sequence. To elucidate the role of the metal ion, the reactions of the EcoRV restriction enzyme were studied in the presence of MnCl2 instead of MgCl2. The reaction at the EcoRV recognition site was slower with Mn2+. This was caused partly by reduced rates for phosphodiester hydrolysis but also by the translocation of the enzyme along the DNA after cleaving it in one strand. In contrast, alternative sites that differ from the recognition site by one base pair were cleaved faster in the presence of Mn2+ relative to Mg2+. When located at an alternative site on the DNA, the EcoRV enzyme bound Mn2+ ions readily but had a very low affinity for Mg2+. The EcoRV nuclease is thus restrained from cleaving DNA at alternate sites in the presence of Mg2+, but the restraint fails to operate with Mn2+. A discrimination factor, which measures the ratio of the activity of the EcoRV nuclease at its recognition site over that at an alternative site, had values of 3 x 10(5) in MgCl2 and 6 in MnCl2.     

Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.

The rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.     

Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease.

Measurements of binding equilibria of EcoRV endonuclease to DNA, for a series of base-analogue substrates, demonstrate that expression of sequence selectivity is strongly enhanced by the presence of Ca2+ ions. Binding constants were determined for short duplex oligodeoxynucleotides containing the cognate DNA site, three cleavable noncognate sites, and a fully nonspecific site. At pH 7.5 and 100 mM NaCl, the full range of specificity from the specific (tightest binding) to nonspecific (weakest binding) sites is 0.9 kcal/mol in the absence of metal ions and 5.8 kcal/mol in the presence of Ca2+. Precise determination of binding affinities in the presence of the active Mg2+ cofactor was found to be possible for substrates retaining up to 1.6% of wild-type activity, as determined by the rate of phosphoryl transfer. These measurements show that Ca2+ is a near-perfect analogue for Mg2+ in binding reactions of the wild-type enzyme with DNA base-analogue substrates, as it provides identical DeltaDeltaG degrees bind values among the cleavable noncognate sites. Equilibrium dissociation constants of wild-type and base-analogue sites were also measured for the weakly active EcoRV mutant K38A, in the presence of either Mg2+ or Ca2+. In this case, Ca2+ allows expression of a greater degree of specificity than does Mg2+. DeltaDeltaG degrees bind values of K38A toward specific versus nonspecific sites are 6.1 kcal/mol with Ca2+ and 3.9 kcal/mol with Mg2+, perhaps reflecting metal-specific conformational changes in the ground-state ternary complexes. The enhancement of binding specificity provided by divalent metal ions is likely to be general to many restriction endonucleases and other metal-dependent nucleic acid-modifying enzymes. These results strongly suggest that measurements of DNA binding affinities for EcoRV, and likely for many other restriction endonucleases, should be performed in the presence of divalent metal ions.     

Mg2+ confers DNA binding specificity to the EcoRV restriction endonuclease.

The EcoRV mutant D90A which carries an amino acid substitution in its active center does not cleave DNA. Therefore, it is possible to perform DNA binding experiments with the EcoRV-D90A mutant both in the absence and in the presence of Mg2+. Like wild-type EcoRV [Taylor et al. (1991) Biochemistry 30, 8743-8753], it does not show a pronounced specificity for binding to its recognition site in the absence of Mg2+ as judged by the appearance of multiple shifted bands in an electrophoretic mobility shift assay with a 377-bp DNA fragment carrying a single EcoRV recognition sequence. In the presence of Mg2+, however, only one band corresponding to a 1:1 complex appears even with a high excess of protein over DNA. This complex most likely is the specific one, because its formation is suppressed much more effectively by a 13-bp oligodeoxynucleotide with an EcoRV site than by a corresponding oligodeoxynucleotide without an EcoRV site. The preferential interaction of the EcoRV-D90A mutant with specific DNA in the presence of Mg2+ was also demonstrated directly: a 20-bp oligodeoxynucleotide with an EcoRV site is bound with KAss = 4 x 10(8) M-1, while a corresponding oligodeoxynucleotide without an EcoRV site is bound with KAss less than or equal to 1 x 10(5) M-1. From these data it appears that Mg2+ confers DNA binding specificity to this mutant by lowering the affinity to nonspecific sites and raising the affinity to specific sites as compared to binding in the absence of Mg2+. It is concluded that this is also true for wild-type EcoRV.     

Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis.

Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.     

Exploring the catalytic center of TaqI endonuclease: rescuing catalytic activity by double mutations and Mn2+

TaqI is a metal-dependent endonuclease that recognizes T(downward arrow)CGA, with the arrow indicating the cleavage site. Mutations at K158 render the enzyme inactive and mutations at K157 significantly reduce DNA cleavage activity (W. Cao and F. Barany (1998) J. Biol. Chem. 273, 33002-33010). Aspartate, glutamate, and histidine substitutions were made at K158 in the wild-type and K157S mutant TaqI endonuclease to understand the functional organization of the active site. None of the mutants was active with Mg(2+), but the DNA cleavage activities were partly rescued by Mn2+ for K157S-K158E and K157S-K158H mutants. The rescuing effects were observed with Mn2+ but not with other divalent cations. K157S-K158E required higher Mn2+ concentrations than the wild-type enzyme for DNA cleavage activity, suggesting that a Mn2+ ion is weakly bound at the 158 position. The need to neutralize K157 to recover the catalytic activity of K158E and K158H indicates that K158 and K157 may interact functionally. In analogy with EcoRV, Ca2+ stimulated Mn2+-mediated cleavage for the wild-type TaqI, suggesting the existence of at least two metal ions at the catalytic center. A catalytic mechanism involving two metal ions and the K157-K158 pair is proposed for TaqI endonuclease.     

Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 10(3)-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2+ in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherichia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2+ as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metal preference as well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.     

Modes of DNA cleavage by the EcoRV restriction endonuclease

The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.     

Electrostatic contributions to site specific DNA cleavage by EcoRV endonuclease

Mutational analysis of amino acids at the periphery of the EcoRV endonuclease active site suggests that moderate-range electrostatic effects play a significant role in modulating the efficiency of phosphoryl transfer. Asp36 and Lys38 located on minor-groove binding surface loops approach within 7-9 A of the scissile phosphates of the DNA. While the rates of single-site mutations removing the carboxylate or amine moieties at these positions are decreased 10(3)-10(5)-fold compared to that of wild-type EcoRV, we find that double mutants which rebalance the charge improve catalysis by up to 500-fold. Mutational analysis also suggests that catalytic efficiency is influenced by Lys173, which is buried at the base of a deep depression penetrating from a distal surface of the enzyme. The Lys173 amine group lies just 6 A from the amine group of the conserved essential Lys92 side chain in the active site. Kinetic and crystallographic analyses of the EcoRV E45A mutant enzyme further show that the Glu45 carboxylate group facilitates an extensive set of conformational transitions which occur upon DNA binding. The crystal structure of E45A bound to DNA and Mn2+ ions reveals significant conformational alterations in a small alpha-helical portion of the dimer interface located adjacent to the DNA minor groove. This leads to a tertiary reorientation of the two monomers as well as shifting of the key major-groove binding recognition loops. Because the Glu45 side chain does not appear to play a direct structural role in maintaining the active site, these rearrangements may instead originate in an altered electrostatic potential caused by removal of the negative charge. A Mn2+ binding site on the scissile phosphate is also disrupted in the E45A structure such that inner-sphere metal interactions made by the scissile DNA phosphate and conserved Asp90 carboxylate are each replaced with water molecules in the mutant. These findings argue against a proposed role for Asp36 as the general base in EcoRV catalysis, and reveal that the induced-fit conformational changes necessary for active site assembly and metal binding are significantly modulated by the electrostatic potential in this region.     

Temperature-sensitive mutant of Bacillus subtilis glutamine synthetase obtained by random mutation

Random mutations were introduced into the B. subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected. DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe. The mutant enzyme was purified, and its kinetic and physical properties were characterized. The Mg2(+)-dependent activity and Mg2+ plus Mn2(+)-dependent activity of the mutant were less than 5% of those of the wild-type at 37 degrees C, and these activities decreased above 15 degrees C, whereas the Mn2(+)-dependent activity was nearly normal. Affinity of the mutant enzyme for glutamate was extremely decreased although the Km values for NH3 or ATP were almost the same as those of the wild-type. The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg2+, although addition of glutamate, ATP, and Mn2+ completely protected both enzymes. These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg2+.     

Binding kinetics and footprinting of TaqI endonuclease: effects of metal cofactors on sequence-specific interactions.

Restriction endonucleases achieve sequence-specific recognition and strand cleavage through the interplay of base, phosphate backbone, and metal cofactor interactions. In this study, we investigate the binding kinetics of TaqI endonuclease using the wild-type enzyme and a binding proficient, catalysis deficient mutant TaqI-D137A both in the absence of a metal cofactor and in the presence of Mg2+ or Ca2+. As demonstrated by gel mobility shift analyses, TaqI endonuclease requires a metal cofactor for achieving high-affinity specific binding to its cognate sequence, TCGA. In the absence of a metal cofactor, the enzyme binds all DNA sequences (TaqI cognate site, star site, and nonspecific site) with essentially equal affinity, thereby exhibiting little discrimination. The dissociation constant of the cognate sequence in the presence of Mg2+ at 60 degrees C is 0. 26 nM, a value comparable to our previously reported Km of 0.5 nM measured under steady-state conditions. The TaqI-TCGA-Mg2+ complex is stable, with a half-life of 21 min at 60 degrees C. The boundary of the protein-DNA interface is approximated to be about 18 bp as determined by DNase I footprinting. Data from this study support the notion that a metal cofactor plays a critical role for achieving sequence-specific discrimination in a subset of nucleases, including TaqI, EcoRV, and others.     

EcoRV restrictase: physical and catalytic properties of homogenous enzyme

With the use of the strain-overproducer restriction endonuclease R.EcoRV was isolated and purified to homogeneity. The molecular mass of the enzyme was determined by gel filtration and polyacrylamide gel electrophoresis to be 25 000 daltons. According to the data of immunological tests R.EcoRV differs in its antigenic characteristics from restriction endonucleases R.EcoRI and R.EcoRII. Dependence of enzyme activity on pH, ionic strength, temperature, presence of divalent cations (Mn2+, Mg2+, Co2+, Zn2+, Ni2+ and Cd2+) and organic solvents (glycerol, dimethylsulfoxide, ethanol) has been studied. It was shown that under conditions of replacement of Mg2+ for Mn2+ or after addition of organic solvents relaxation of R.EcoRV specificity takes place. It was shown also that R.EcoRV is able to digest T-even bacteriophage DNAs with different types and extents of modification. DNA modified by the action of MR.EcoRV system in vivo is susceptible to R.EcoRV in vitro. Under conditions of relaxed specificity noncanonical sites are susceptible to R.EcoRV attack. The fragments resulted may be cloned in canonical pBR322 EcoRV site.     

Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage

The carboxy-terminal subdomains of the homodimeric EcoRV restriction endonuclease each bear a net charge of +4 and are positioned on the inner concave surface of the 50 degree DNA bend that is induced by the enzyme. A complete kinetic and structural analysis of a truncated EcoRV mutant lacking these domains was performed to assess the importance of this diffuse charge in facilitating DNA binding, bending, and cleavage. At the level of formation of an enzyme-DNA complex, the association rate for the dimeric mutant enzyme was sharply decreased by 10(3)-fold, while the equilibrium dissociation constant was weakened by nearly 10(6)-fold compared with that of wild-type EcoRV. Thus, the C-terminal subdomains strongly stabilize the enzyme-DNA ground-state complex in which the DNA is known to be bent. Further, the extent of DNA bending as observed by fluorescence resonance energy transfer was also significantly decreased. The crystal structure of the truncated enzyme bound to DNA and calcium ions at 2.4 A resolution reveals that the global fold is preserved and suggests that a divalent metal ion crucial to catalysis is destabilized in the active site. This may explain the 100-fold decrease in the rate of metal-dependent phosphoryl transfer observed for the mutant. These results show that diffuse positive charge associated with the C-terminal subdomains of EcoRV plays a key role in DNA association, bending, and cleavage.     

EcoRV restriction endonuclease binds all DNA sequences with equal affinity

In the presence of MgCl2, the EcoRV restriction endonuclease cleaves its recognition sequence on DNA at least a million times more readily than any other sequence. In this study, the binding of the EcoRV restriction enzyme to DNA was examined in the absence of Mg2+. With each DNA fragment tested, several DNA-protein complexes were detected by electrophoresis through polyacrylamide. No differences were observed between isogenic DNA molecules that either contained or lacked the EcoRV recognition site. The number of complexes with each fragment varied with the length of the DNA. Three complexes were formed with a DNA molecule of 55 base pairs, corresponding to the DNA bound to 1, 2, or 3 molecules of the protein, while greater than 15 complexes were formed with a DNA of 381 base pairs. A new method was developed to analyze the binding of a protein to multiple sites on DNA. The method showed that the EcoRV enzyme binds to all DNA sequences, including the EcoRV recognition site, with the same equilibrium constant, though two molecules of the protein bind preferentially to adjacent sites on the DNA in a cooperative fashion. All of the complexes with a substrate that contained the EcoRV site dissociated upon addition of competitor DNA, but when the competitor was mixed with MgCl2, a fraction of the substrate was cleaved at the EcoRV site. The fraction cleaved was due mainly to the translocation of the enzyme from nonspecific sites on the DNA to the specific site.     

Discrimination between DNA sequences by the EcoRV restriction endonuclease

The EcoRV restriction endonuclease cleaves not only its recognition sequence on DNA, GATATC, but also, at vastly reduced rates, a number of alternative DNA sequences. The plasmid pAT153 contains 12 alternative sites, each of which differs from the recognition sequence by one base pair. The EcoRV nuclease showed a marked preference for one particular site from among these alternatives. This noncognate site was located at the sequence GTTATC, and the mechanism of action of EcoRV at this site was analyzed. The mechanism differed from that at the cognate site in three respects. First, the affinity of the enzyme for the noncognate site was lower than that for the cognate site, but, by itself, this cannot account for the specificity of EcoRV as measured from the values of kcat/Km. Second, the enzyme had a lower affinity for Mg2+ when it was bound to the noncognate site than when it was bound to its cognate site: this appears to be a key factor in limiting the rates of DNA cleavage at alternative sites. Third, the reaction pathway at the noncognate site differed from that at the cognate site. At the former, the EcoRV enzyme cleaved first one strand of the DNA and then the other while at the latter, both strands were cut in one concerted reaction. The difference in reaction pathway allows DNA ligase to proofread the activity of EcoRV by selective repair of single-strand breaks at noncognate sites, as opposed to double-strand breaks at the cognate site. The addition of DNA ligase to reactions with EcoRV made no difference to product formation at the cognate site, but products from reactions at noncognate sites were no longer detected.     

The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning.

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition.     

DNA cleavage by the EcoRV restriction endonuclease: pH dependence and proton transfers in catalysis.

To characterise the pH dependence of phosphodiester hydrolysis by the EcoRV endonuclease in the presence of Mn2+, single turnover reactions on a 12 bp DNA substrate were examined by stopped-flow and quench-flow methods between pH 6.0 and 8.5. At each pH value, the apparent rate constants for phosphodiester hydrolysis increased hyperbolically with the concentration of MnCl2, thus allowing values to be determined for the intrinsic rate constant at saturation with Mn2+ and the equilibrium dissociation constant for Mn2+. The equilibrium constants showed no systematic variation across the pH range tested, while the rate constants increased steeply with increasing pH up to an asymptote above pH 7.5. At low pH conditions, the gradient of a plot of log (rate constant) against pH approached a value of 2. DNA cleavage by EcoRV thus requires the de-protonation of two acidic groups. To determine whether aspartate 36 is one of the groups, mutants of EcoRV were made with other amino acid residues at position 36. Glutamate caused a partial loss of activity, while all other replacements gave near-zero activities. In contrast to wild-type EcoRV, the mutant with glutamate required the de-protonation of only one acidic group for DNA cleavage. A mechanism for EcoRV is proposed in which the water molecule that hydrolyses the phosphodiester bond is de-protonated by two Bronsted bases, probably the ionised forms of aspartate 36 and glutamate 45.     

Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.     

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)     

Location of the coenzyme binding site in the porcine mitochondrial NADP-dependent isocitrate dehydrogenase

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.