A single mismatch in the DNA induces enhanced aggregation of MutS. Hydrodynamic analyses of the protein-DNA complexes

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10-12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18-20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh > 500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPgammaS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh > 500 nm in the presence of MutL) as compared to the normal homoduplex (Rh approximately 100-200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system.     

Modulation of MutS ATP hydrolysis by DNA cofactors

Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics. At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM. Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM. The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs. The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair. Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis. Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate. In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis. Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly. These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding.     

Crystal structure and biochemical analysis of the MutS.ADP.beryllium fluoride complex suggests a conserved mechanism for ATP interactions in mismatch repair

During mismatch repair ATP binding and hydrolysis activities by the MutS family proteins are important for both mismatch recognition and for transducing mismatch recognition signals to downstream repair factors. Despite intensive efforts, a MutS.ATP.DNA complex has eluded crystallographic analysis. Searching for ATP analogs that strongly bound to Thermus aquaticus (Taq) MutS, we found that ADP.beryllium fluoride (ABF), acted as a strong inhibitor of several MutS family ATPases. Furthermore, ABF promoted the formation of a ternary complex containing the Saccharomyces cerevisiae MSH2.MSH6 and MLH1.PMS1 proteins bound to mismatch DNA but did not promote dissociation of MSH2.MSH6 from mismatch DNA. Crystallographic analysis of the Taq MutS.DNA.ABF complex indicated that although this complex was very similar to that of MutS.DNA.ADP, both ADP.Mg(2+) moieties in the MutS. DNA.ADP structure were replaced by ABF. Furthermore, a disordered region near the ATP-binding pocket in the MutS B subunit became traceable, whereas the equivalent region in the A subunit that interacts with the mismatched nucleotide remained disordered. Finally, the DNA binding domains of MutS together with the mismatched DNA were shifted upon binding of ABF. We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the ordered loop and Domain III in addition to the intra-subunit helical lever arm that links the ATPase and DNA binding domains.     

Interaction of Escherichia coli MutS and MutL at a DNA mismatch

MutS and MutL are both required to activate downstream events in DNA mismatch repair. We examined the rate of dissociation of MutS from a mismatch using linear heteroduplex DNAs or heteroduplexes blocked at one or both ends by four-way DNA junctions in the presence and absence of MutL. In the presence of ATP, dissociation of MutS from linear heteroduplexes or heteroduplexes blocked at only one end occurs within 15 s. When both duplex ends are blocked, MutS remains associated with the DNA in complexes with half-lives of 30 min. DNase I footprinting of MutS complexes is consistent with migration of MutS throughout the DNA duplex region. When MutL is present, it associates with MutS and prevents ATP-dependent migration away from the mismatch in a manner that is dependent on the length of the heteroduplex. The rate and extent of mismatch-provoked cleavage at hemimethylated GATC sites by MutH in the presence of MutS, MutL, and ATP are the same whether the mismatch and GATC sites are in cis or in trans. These results suggest that a MutS-MutL complex in the vicinity of a mismatch is involved in activating MutH.     

Differential and simultaneous adenosine di- and triphosphate binding by MutS

The roles of ATP binding and hydrolysis in the function of MutS in mismatch repair are poorly understood. As one means of addressing this question, we have determined the affinities and number of adenosine di- and triphosphate binding sites within MutS. Nitrocellulose filter binding assay and equilibrium fluorescence anisotropy measurements have demonstrated that MutS has one high affinity binding site for ADP and one high affinity site for nonhydrolyzable ATP analogues per dimer equivalent. Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) promote ADP binding and a large excess of AMPPNP is required to displace ADP from the protein. Fluorescence energy transfer and filter binding assays indicate that ADP and nonhydrolyzable ATP analogues can bind simultaneously to adjacent subunits within the MutS oligomer with affinities in the low micromolar range. These findings suggest that the protein exists primarily as the ATP.MutS.ADP ternary complex in solution and that this may be the form of the protein that is involved in DNA encounters in vivo.     

Mechanism for verification of mismatched and homoduplex DNAs by nucleotides‐bound MutS analyzed by molecular dynamics simulations

In order to understand how MutS recognizes mismatched DNA and induces the reaction of DNA repair using ATP, the dynamics of the complexes of MutS (bound to the ADP and ATP nucleotides, or not) and DNA (with mismatched and matched base‐pairs) were investigated using molecular dynamics simulations. As for DNA, the structure of the base‐pairs of the homoduplex DNA which interacted with the DNA recognition site of MutS was intermittently disturbed, indicating that the homoduplex DNA was unstable. As for MutS, the disordered loops in the ATPase domains, which are considered to be necessary for the induction of DNA repair, were close to (away from) the nucleotide‐binding sites in the ATPase domains when the nucleotides were (not) bound to MutS. This indicates that the ATPase domains changed their structural stability upon ATP binding using the disordered loop. Conformational analysis by principal component analysis showed that the nucleotide binding changed modes which have structurally solid ATPase domains and the large bending motion of the DNA from higher to lower frequencies. In the MutS–mismatched DNA complex bound to two nucleotides, the bending motion of the DNA at low frequency modes may play a role in triggering the formation of the sliding clamp for the following DNA‐repair reaction step. Moreover, MM‐PBSA/GBSA showed that the MutS–homoduplex DNA complex bound to two nucleotides was unstable because of the unfavorable interactions between MutS and DNA. This would trigger the ATP hydrolysis or separation of MutS and DNA to continue searching for mismatch base‐pairs. Proteins 2016; 84:1287–1303. © 2016 Wiley Periodicals, Inc.     

Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex

The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood. MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W. (2001) Mol. Cell 7, 1-12). Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A. In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08-0.58 mol/dimer), consistent with the suggestion that the ADP.MutS.ATP complex comprises a significant fraction of the protein in solution (Bjornson, K. P. and Modrich, P. (2003) J. Biol. Chem. 278, 18557-18562). In the presence of Mg2+, endogenous ATP is hydrolyzed with a rate constant of 0.12 min-1 at 30 degrees C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA. As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch. However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS. The mutant protein also fails to support normal assembly of the MutS.MutL.DNA ternary complex. Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.     

The role of nucleotide cofactor binding in cooperativity and specificity of MutS recognition

Mismatch repair (MMR) is essential for eliminating biosynthetic errors generated during replication or genetic recombination in virtually all organisms. The critical first step in Escherichia coli MMR is the specific recognition and binding of MutS to a heteroduplex, containing either a mismatch or an insertion/deletion loop of up to four nucleotides. All known MutS homologs recognize a similar broad spectrum of substrates. Binding and hydrolysis of nucleotide cofactors by the MutS-heteroduplex complex are required for downstream MMR activity, although the exact role of the nucleotide cofactors is less clear. Here, we showed that MutS bound to a 30-bp heteroduplex containing an unpaired T with a binding affinity ≈ 400-fold stronger than to a 30-bp homoduplex, a much higher specificity than previously reported. The binding of nucleotide cofactors decreased both MutS specific and nonspecific binding affinity, with the latter marked by a larger drop, further increasing MutS specificity by ≈ 3-fold. Kinetic studies showed that the difference in MutS K_d for various heteroduplexes was attributable to the difference in intrinsic dissociation rate of a particular MutS-heteroduplex complex. Furthermore, the kinetic association event of MutS binding to heteroduplexes was marked by positive cooperativity. Our studies showed that the positive cooperativity in MutS binding was modulated by the binding of nucleotide cofactors. The binding of nucleotide cofactors transformed E. coli MutS tetramers, the functional unit in E. coli MMR, from a cooperative to a noncooperative binding form. Finally, we found that E. coli MutS bound to single-strand DNA with significant affinity, which could have important implication for strand discrimination in eukaryotic MMR mechanism.     

Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA

Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2-Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30-50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1(ADP)-S2(ATP) is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1(ATP)-S2(ATP) and S1(ATP)-S2(ADP) are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair.     

Differential specificities and simultaneous occupancy of human MutSalpha nucleotide binding sites

We have examined the permissible nucleotide occupancy states of human MutSalpha. The MSH2.MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5'-O-(thiotriphosphate) (ATPgammaS), with a K(d) for each nucleotide of about 1 microm. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutSalpha heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPgammaS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutSalpha is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutSalpha-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutSalpha.heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutSalpha and ADP or a triphosphate analogue. These findings imply that MutSalpha has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP.MutSalpha.ATP ternary complex has an important role in mismatch repair.     

ATP-hydrolysis-dependent conformational switch modulates the stability of MutS-mismatch complexes

The mismatch repair pathway in Escherichia coli has been extensively studied in vitro as well as in vivo. The molecular mechanisms by which nucleotide cofactors regulate the whole process constitute an area of active debate. Here we demonstrate that nucleotide (ADP or ATP) binding to MutS mediates a switch in protein conformation. However, in MutS that is DNA bound, this switch ensues only with ATP and not with ADP and is similar, irrespective of whether it is bound to a homo- or a heteroduplex. The results envisage a minimal model of three conformational states of MutS as reflected in: (i) a specific and highly stable MutS–mismatch complex in the absence of a nucleotide; (ii) a specific but less stable complex in the presence of ATP hydrolysis; and (iii) an irreversibly dissociated complex in the presence of ATP binding (ATPγS). Such transitions are of relevance to the protein’s function in vivo where it has to first recognize a mismatch, followed by a search for hemimethylated sites.     

Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities.

The Salmonella typhimurium and Escherichia coli MutS protein is one of several methyl-directed mismatch repair proteins that act together to correct replication errors. MutS is homologous to the Streptococcus pneumoniae HexA mismatch repair protein and to the Duc1 and Rep1 proteins of human and mouse. Homology between the deduced amino acid sequence of both MutS and HexA, and the type A nucleotide binding site consensus sequence, suggested that ATP binding and hydrolysis play a role in their mismatch repair functions. We found that MutS does indeed weakly hydrolyze ATP to ADP and Pi, with a Km of 6 microM and kcat of 0.26. To show that this activity is intrinsic to MutS, we made a site-directed mutation, which resulted in the invariant lysine of the nucleotide binding consensus sequence being changed to an alanine. The mutant MutS allele was unable to complement a mutS::Tn10 mutation in vivo, and was dominant over wild type when present in high copy number. The purified mutant protein had reduced ATPase activity, with the Km affected more severely than the kcat. Like the wild type MutS protein, the mutant protein is able to bind heteroduplex DNA specifically, but the mutant protein does so with a reduced affinity.     

Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSalpha is abolished by a single mutation in the hMSH6 subunit

The most abundant mismatch binding factor in human cells, hMutSalpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein. The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., Camerini-Otero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutSalpha. The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutSalpha were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutSalpha not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA.     

Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling

MutS protein recognizes mispaired bases in DNA and targets them for mismatch repair. Little is known about the transient conformations of MutS as it signals initiation of repair. We have used single-molecule fluorescence resonance energy transfer (FRET) measurements to report the conformational dynamics of MutS during this process. We find that the DNA-binding domains of MutS dynamically interconvert among multiple conformations when the protein is free and while it scans homoduplex DNA. Mismatch recognition restricts MutS conformation to a single state. Steady-state measurements in the presence of nucleotides suggest that both ATP and ADP must be bound to MutS during its conversion to a sliding clamp form that signals repair. The transition from mismatch recognition to the sliding clamp occurs via two sequential conformational changes. These intermediate conformations of the MutS:DNA complex persist for seconds, providing ample opportunity for interaction with downstream proteins required for repair.     

Formation of a DNA mismatch repair complex mediated by ATP

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.     

Functional characterization of the DNA mismatch binding protein MutS from Haemophilus influenzae

This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein-DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process.     

Self-assembly of Escherichia coli MutL and its complexes with DNA

The Escherichia coli MutL protein regulates the activity of several enzymes, including MutS, MutH, and UvrD, during methyl-directed mismatch repair of DNA. We have investigated the self-association properties of MutL and its binding to DNA using analytical sedimentation velocity and equilibrium. Self-association of MutL is quite sensitive to solution conditions. At 25 °C in Tris at pH 8.3, MutL assembles into a heterogeneous mixture of large multimers. In the presence of potassium phosphate at pH 7.4, MutL forms primarily stable dimers, with the higher-order assembly states suppressed. The weight-average sedimentation coefficient of the MutL dimer in this buffer ( ?s(20,w)) is equal to 5.20 ± 0.08 S, suggesting a highly asymmetric dimer (f/f(o) = 1.58 ± 0.02). Upon binding the nonhydrolyzable ATP analogue, AMPPNP/Mg(2+), the MutL dimer becomes more compact ( ?s(20,w) = 5.71 ± 0.08 S; f/f(o) = 1.45 ± 0.02), probably reflecting reorganization of the N-terminal ATPase domains. A MutL dimer binds to an 18 bp duplex with a 3'-(dT(20)) single-stranded flanking region, with apparent affinity in the micromolar range. AMPPNP binding to MutL increases its affinity for DNA by a factor of ～10. These results indicate that the presence of phosphate minimizes further MutL oligomerization beyond a dimer and that differences in solution conditions likely explain apparent discrepancies in previous studies of MutL assembly.     

ATP increases the affinity between MutS ATPase domains. Implications for ATP hydrolysis and conformational changes

MutS is the key protein of the Escherichia coli DNA mismatch repair system. It recognizes mispaired and unpaired bases and has intrinsic ATPase activity. ATP binding after mismatch recognition by MutS serves as a switch that enables MutL binding and the subsequent initiation of mismatch repair. However, the mechanism of this switch is poorly understood. We have investigated the effects of ATP binding on the MutS structure. Crystallographic studies of ATP-soaked crystals of MutS show a trapped intermediate, with ATP in the nucleotide-binding site. Local rearrangements of several residues around the nucleotide-binding site suggest a movement of the two ATPase domains of the MutS dimer toward each other. Analytical ultracentrifugation experiments confirm such a rearrangement, showing increased affinity between the ATPase domains upon ATP binding and decreased affinity in the presence of ADP. Mutations of specific residues in the nucleotide-binding domain reduce the dimer affinity of the ATPase domains. In addition, ATP-induced release of DNA is strongly reduced in these mutants, suggesting that the two activities are coupled. Hence, it seems plausible that modulation of the affinity between ATPase domains is the driving force for conformational changes in the MutS dimer. These changes are driven by distinct amino acids in the nucleotide-binding site and form the basis for long-range interactions between the ATPase domains and DNA-binding domains and subsequent binding of MutL and initiation of mismatch repair.     

Binding of magnesium to myosin subfragment-1 ATPase

Tyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylylimidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435 nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of delta A at 435 nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25 X 10(-8) M and 1.24 X 10(-7) with AMPPNP and ADP, respectively. The difference spectrum was also obtained in the presence of ATP. The delta epsilon value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADP(a), was estimated as 6 X 10(-8) M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.     

Rapid filtration analysis of nucleotide binding to Na,K-ATPase

Transient kinetic analysis of nucleotide binding to pig kidney Na,K-ATPase using a rapid filtration technique shows that the interaction between nucleotide and enzyme apparently follows simple first-order kinetics both for ATP in the absence of Mg(2+) and for ADP in the presence or absence of Mg(2+). Rapid filtration experiments with Na,K-ATPase membrane sheets may nevertheless suffer from a problem of accessibility for a fraction of the ATPase binding sites. Accordingly, we estimate from these data that for ATP binding in the absence of Mg(2+) and the presence of 35 mM Na(+) at pH 7.0 at 20 degrees C, the bimolecular binding rate constant k(on) is about 30 microM(-1) x s(-1) and the dissociation rate constant k(off) is about 8 s(-1). In the presence of 10 mM Mg(2+), the binding rate constant is the same as that in the absence of Mg(2+). For ADP or MgADP the binding rate constant is about 20 microM(-1) x s(-1) and the dissociation rate constant is about 12 s(-1). Results of rapid-mixing stopped-flow experiments with the fluorescent dye eosin are also consistent with a one-step mechanism of binding of eosin to the ATPase nucleotide site. The implication of these results is that nucleotide binding to Na,K-ATPase both in the absence and presence of Mg(2+) appears to be a single-step event, at least on the time scale accessible in these experiments.