One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single-turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of the metallonuclease mechanism. DNA association rate constants are approximately 100-fold higher in the presence of the catalytically nonsupportive Ca(II) versus the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM(2)S and EM(4)S, respectively) support cleavage. Interestingly, the cleavage rate for EM(2)S is approximately 100-fold slower than that displayed by EM(4)S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two-metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.     

Crystal structure of monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8

Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.     

Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.     

Metal-dependent DNA cleavage mechanism of the I-CreI LAGLIDADG homing endonuclease

The LAGLIDADG homing endonucleases include free-standing homodimers, pseudosymmetric monomers, and related enzyme domains embedded within inteins. DNA-bound structures of homodimeric I-CreI and monomeric I-SceI indicate that three catalytic divalent metal ions are distributed across a pair of overlapping active sites, with one shared metal participating in both strand cleavage reactions. These structures differ in the precise position and binding interactions of the metals. We have studied the metal dependence for the I-CreI homodimer using site-directed mutagenesis of active site residues and assays of binding affinity and cleavage activity. We have also reassessed the binding of a nonactivating metal ion (calcium) in the wild-type enzyme-substrate complex, and determined the DNA-bound structure of two inactive enzyme mutants. The conclusion of these studies is that the catalytic mechanism of symmetric LAGLIDADG homing endonucleases, and probably many of their monomeric cousins, involves a canonical two-metal mechanism in each of two active sites, which are chemically and structurally tethered to one another by a shared metal ion. Failure to occupy the shared metal site, as observed in the presence of calcium or when the metal-binding side chain from the LAGLIDADG motif (Asp 20) is mutated to asparagine, prevents cleavage by the enzyme.     

Roles of divalent metal ions in flap endonuclease-substrate interactions

Flap endonucleases (FENs) have essential roles in DNA processing. They catalyze exonucleolytic and structure-specific endonucleolytic DNA cleavage reactions. Divalent metal ions are essential cofactors in both reactions. The crystal structure of FEN shows that the protein has two conserved metal-binding sites. Mutations in site I caused complete loss of catalytic activity. Mutation of crucial aspartates in site II abolished exonuclease action, but caused enzymes to retain structure-specific (flap endonuclease) activity. Isothermal titration calorimetry revealed that site I has a 30-fold higher affinity for cofactor than site II. Structure-specific endonuclease activity requires binding of a single metal ion in the high-affinity site, whereas exonuclease activity requires that both the high- and low-affinity sites be occupied by divalent cofactor. The data suggest that a novel two-metal mechanism operates in the FEN-catalyzed exonucleolytic reaction. These results raise the possibility that local concentrations of free cofactor could influence the endo- or exonucleolytic pathway in vivo.     

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis

To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation. Here, we test global kinetic analysis as an approach to this problem. Taking advantage of the various binding and cleavage constants established for PvuII endonuclease, we apply cleavage data obtained under a range of Mg(II) and Ca(II) concentrations to a number of kinetic models which specify A and B sites for both metal ions and various active species. The data are best fit and simulated with models which feature Ca(II) being held more strongly in the B (or secondary) site. This mixed metal enzyme species is the only one which forms appreciably and exhibits a cleavage rate constant similar to that observed when there is only one Mg(II) per active site (approximately 0.01 s^?1). Thus, in the case of PvuII endonuclease, Ca(II) does not stimulate cleavage. However, a simulated increase in activity at moderate Ca(II) concentrations can be rationalized with a cleavage rate constant for the mixed species similar to that when two Mg(II) ions are present in the active site. This provides an important insight into the underlying basis for the Ca(II)-stimulated activity observed for some metallonucleases that is not accessible by any other means.     

A "moving metal mechanism" for substrate cleavage by the DNA repair endonuclease APE-1

Apurinic/apyrimidinic endonuclease (APE-1) is essential for base excision repair (BER) of damaged DNA. Here molecular dynamics (MD) simulations of APE1 complexed with cleaved and uncleaved damaged DNA were used to determine the role and position of the metal ion(s) in the active site before and after DNA cleavage. The simulations started from an energy minimized wild-type structure of the metal-free APE1/damaged-DNA complex (1DE8). A grid search with one Mg2+ ion located two low energy clusters of Mg2+ consistent with the experimentally determined metal ion positions. At the start of the longer MD simulations, Mg2+ ions were placed at different positions as seen in the crystal structures and the movement of the ion was followed over the course of the trajectory. Our analysis suggests a "moving metal mechanism" in which one Mg2+ ion moves from the B- (more buried) to the A-site during substrate cleavage. The anticipated inversion of the phosphate oxygens occurs during the in-line cleavage reaction. Experimental results, which show competition between Ca2+ and Mg2+ for catalyzing the reaction, and high concentrations of Mg2+ are inhibitory, indicate that both sites cannot be simultaneously occupied for maximal activity.     

Metal ion interaction with cosubstrate in self-splicing of group I introns.

The catalytic mechanism for self-splicing of the group I intron in the pre-mRNA from the nrdB gene in bacteriophage T4 has been investigated using 2'-amino- 2'-deoxyguanosine or guanosine as cosubstrates in the presence of Mg2+, Mn2+and Zn2+. The results show that a divalent metal ion interacts with the cosubstrate and thereby influences the efficiency of catalysis in the first step of splicing. This suggests the existence of a metal ion that catalyses the nucleophilic attack of the cosubstrate. Of particular significance is that the transesterification reactions of the first step of splicing with 2'-amino-2'-deoxyguanosine as cosubstrate are more efficient in mixtures containing either Mn2+or Zn2+together with Mg2+than with only magnesium ions present. The experiments in metal ion mixtures show that two (or more) metal ions are crucial for the self-splicing of group I introns and suggest the possibility that more than one of these have a direct catalytic role. A working model for a two-metal-ion mechanism in the transesterification steps is suggested.     

Iron and copper chelation by flavonoids: an electrospray mass spectrometry study

Flavonoids are well known as effective free radical scavengers exhibiting therefore an antioxidant behaviour. Another antioxidant mechanism however may result from the ability they have to chelate metal ions, rendering them inactive to participate in free radical generating reactions. Electrospray mass spectrometry has been used to study metal ion interactions with a set of flavonoids from different classes. Complexes with a range of stoichiometries, of metal: flavonoid, 1:1, 1:2, 2:2, 2:3 have been observed. The stoichiometry 1:2 is in general the preferred one. It is established for flavones and for the flavanone naringenin that the binding metal sites are preferentially at the 5-hydroxyl and 4-oxo groups. Redox reactions are also observed through the change of the oxidation state of the metal, jointly with the oxidation of the flavonoid by loss of hydrogen. Structures of the oxidized species of some flavonoids are proposed.     

The kinetics of flavine oxidation-reduction. II. Metal ion interactions.

The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.     

Structures of cadmium-binding acidic phospholipase A2 from the venom of Agkistrodon halys Pallas at 1.9A resolution

Phospholipase A(2) coordinates Ca(2+) ion through three carbonyl oxygen atoms of residues 28, 30, and 32, two carboxyl oxygen atoms of residue Asp49, and two (or one) water molecules, forming seven (or six) coordinate geometry of Ca(2+) ligands. Two crystal structures of cadmium-binding acidic phospholipase A(2) from the venom of Agkistrodon halys Pallas (i.e., Agkistrodon blomhoffii brevicaudus) at different pH values (5.9 and 7.4) were determined to 1.9A resolution by the isomorphous difference Fourier method. The well-refined structures revealed that a Cd(2+) ion occupied the position expected for a Ca(2+) ion, and that the substitution of Cd(2+) for Ca(2+) resulted in detectable changes in the metal-binding region: one of the carboxyl oxygen atoms from residue Asp49 was farther from the metal ion while the other one was closer and there were no water molecules coordinating to the metal ion. Thus the Cd(2+)-binding region appears to have four coordinating oxygen ligands. The cadmium binding to the enzyme induced no other significant conformational change in the enzyme molecule elsewhere. The mechanism for divalent cadmium cation to support substrate binding but not catalysis is discussed.     

Identifying the structure of the active sites of human recombinant prolidase

In this paper we provide a detailed biochemical and structural characterization of the active site of recombinant human prolidase, a dimeric metalloenzyme, whose misfunctioning causes a recessive connective tissue disorder (prolidase deficiency) characterized by severe skin lesions, mental retardation and respiratory tract infections. It is known that the protein can host two metal ions in the active site of each constituent monomer. We prove that two different kinds of metals (Mn and Zn) can be simultaneously present in the protein active sites with the protein partially maintaining its enzymatic activity. Structural information extracted from X-ray absorption spectroscopy measurements have been used to yield a full reconstruction of the atomic environment around each one of the two monomeric active sites. In particular, as for the metal ion occupation configuration of the recombinant human prolidase, we have found that one of the two active sites is occupied by two Zn ions and the second one by one Zn and one Mn ion. In both dinuclear units a histidine residue is bound to a Zn ion.     

X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamer.

Metal ion binding to the insulin hexamer has been investigated by crystallographic analysis. Cadmium, lead, and metal-free hexamers have been refined to R values of 0.181, 0.172, and 0.172, against data of 1.9-, 2.5-, and 2.5-A resolution, respectively. These structures have been compared with each other and with the isomorphous two-zinc insulin. The structure of the metal-free hexamer shows that the His(B10) imidazole rings are arranged in a preformed site that binds a water molecule and is poised for Zn2+ coordination. The structure of the cadmium derivative shows that the binding of Cd2+ at the center of the hexamer is unusual. There are three symmetry-related sites located within 2.7 A of each other, and this position is evidently one-third occupied. It is also shown that the coordinating B13 glutamate side chains of this derivative have two partially occupied conformations. One of these conformations is two-thirds occupied and is very similar to that seen in two-zinc insulin. The other, one-third-occupied conformation, is seen to coordinate the one-third-occupied metal ion. The binding of Ca2+ to insulin is assumed to be essentially identical with that of Cd2+. Thus, we conclude that the Ca2+ binding site in the insulin hexamer is unlike that of any other known calcium binding protein. The crystal structures reported herein explain how binding of metal ions stabilizes the insulin hexamer. The role of metal ions in hexamer assembly and dissociation is discussed.     

The molybdenum-cofactor: a crystallographic perspective

 The molybdenum-cofactor (Mo-co) consists of a mononuclear molybdenum or tungsten ion coordinated by one or two molybdopterin ligands. Crystallographic analyses have demonstrated that the molybdopterin ligands are tricyclic and nonplanar, and that they coordinate the metal through their dithiolene sulfurs. Additional ligands to the metal may be provided by amino acid side chains (including serine, cysteine and selenocysteine), as well as one or more nonprotein O or S ligands, such as oxo, hydroxo, and sulfido. The molybdopterin ligand may participate in the various electron transfer reactions associated with the catalytic mechanism of these proteins, as suggested by both oxidation state-dependent changes in the metal coordination environment and the molybdopterin structure, and by the interaction of the molybdopterin with other redox groups within Mo-co-containing enzymes. Received: 14 May 1997 / Accepted: 20 August 1997     

The binding of a second divalent metal ion is necessary for the activation of ATP hydrolysis and its inhibition by tightly bound ADP in the ATPase from Halobacterium saccharovorum.

A binding site for divalent metal ions on the ATPase from Halobacterium saccharovorum is proposed. This site is different from the catalytic site which binds ATP and a complexed divalent metal ion. Occupation of the second site greatly stimulates the rate of ATP hydrolysis and the affinity of the catalytic site for the metal ion-ATP complex. The time-dependent inhibition of the ATPase, which occurs during catalysis and which is known to be caused by the retention of ADP, is also dependent on the occupation of this metal ion binding site. The binding of the metal ion apparently induces extremely tight binding of ADP after the departure of Pi. Mg2+, Mn2+, Zn2+, Co2+, and Ca2+ were tested and showed both the activating and the inhibitory effects, although their binding constants for ATP and the second metal ion binding site were quite different. The characteristic shapes of the nonlinear ATP hydrolysis curves obtained with different metal ions, and different ratios of metal ion and ATP, could be explained with the established dissociation constants. On this basis, a model for the ATPase was developed with two catalytic cycles: one in which the second metal ion binding site is occupied, and another in which it is empty. These pathways are connected by metal ion-dependent equilibria.     

Engineering of the hydrophobic core of an alpha-helical coiled coil

The amino acid sequence that forms the alpha-helical coiled coil structure has a representative heptad repeat denoted by defgabc, according to their positions. Although the a and d positions are usually occupied by hydrophobic residues, hydrophilic residues at these positions sometimes play important roles in natural proteins. We have manipulated a few amino acids at the a and d positions of a de novo designed trimeric coiled coil to confer new functions to the peptides. The IZ peptide, which has four heptad repeats and forms a parallel triple-stranded coiled coil, has Ile at all of the a and d positions. We show three examples: (1) the substitution of one Ile at either the a or d position with Glu caused the peptide to become pH sensitive; (2) the metal ion induced alpha-helical bundles were formed by substitutions with two His residues at the d and a positions for a medium metal ion, and with one Cys residue at the a position for a soft metal ion; and (3) the AAB-type heterotrimeric alpha-helical bundle formation was accomplished by a combination of Ala and Trp residues at the a positions of different peptide chains. Furthermore, we applied these procedures to prepare an ABC-type heterotrimeric alpha-helical bundle and a metal ion-induced heterotrimeric alpha-helical bundle.     

Divalent metal ion differentially regulates the sequential nicking reactions of the GIY-YIG homing endonuclease I-BmoI

Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double-strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conformational changes for reaction progress. Collectively, our data implies mechanistic roles for divalent metal ion and substrate bases, suggesting that divalent metal ion facilitates the re-positioning of the GIY-YIG nuclease domain between sequential nicking reactions.     

A new use for a familiar fold: the X-ray crystal structure of GTP-bound GTP cyclohydrolase III from Methanocaldococcus jannaschii reveals a two metal ion catalytic mechanism

GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg2+ for catalytic activity and is activated by monovalent cations such as K+ and ammonium [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. The reaction is formally identical to that catalyzed by a GCH II ortholog (SCO 6655) from Streptomyces coelicolor; however, SCO 6655, like other GCH II proteins, is a zinc-containing protein. The structure of GCH III complexed with GTP solved at 2 A resolution clearly shows that GCH III adopts a distinct fold that is closely related to the palm domains of phosphodiesterases, such as DNA polymerase I. GCH III is a tetramer of identical subunits; each monomer is composed of an N- and a C-terminal domain that adopt nearly superimposible structures, suggesting that the protein has arisen by gene duplication. Three metal ions were located in the active site, two of which occupy positions that are analogous to those occupied by divalent metal ions in the structures of a number of palm domain containing proteins, such as DNA polymerase I. Two conserved Asp residues that coordinate the metal ions, which are also found in palm domain containing proteins, are observed in GCH III. Site-directed variants (Asp-->Asn) of these residues in GCH III are less active than wild-type. The third metal ion, most likely a potassium ion, is involved in substrate recognition through coordination of O6 of GTP. The arrangement of the metal ions in the active site suggests that GCH III utilizes two metal ion catalysis. The structure of GCH III extends the repertoire of possible reactions with a palm fold to include cyclohydrolase chemistry.     

A study of the coordination shell of aluminum(III) and magnesium(II) in model protein environments: thermodynamics of the complex formation and metal exchange reactions

Al(III) toxicity in living organisms is based on competition with other metal cations. Mg(II) is one of the most affected cations, since the size similarity dominates over the charge identity. The slow ligand exchange rates for Al(III) render the ion useless as a metal ion at the active sites of enzymes and provide a mechanism by which Al(III) inhibits Mg(II) dependent biochemical processes. Al(III) cation interactions with relevant bioligands have been studied in a protein-model environment in gas and aqueous phases using density functional theory methods. The protein model consists of the metal cation bound to two chosen bioligands (functional groups of the amino acid side chains, one of them being always an acetate) and water molecules interacting with the cation to complete its first coordination shell. Analogous Mg(II) complexes are calculated and compared with the Al(III) ones. Formation energies of the complexes are calculated in both phases and magnesium/aluminum exchange energies evaluated. The effect of different dielectric media is also analyzed. The presence of an acetate ligand in the binding site is found to promote both, complex formation and metal exchange reactions. In addition, buried binding sites (with low dielectric constant) of the protein favor metal exchange, whereas fully solvated environments of high dielectric constant require the presence of two anionic ligands for metal exchange to occur.     

Role of metal ions in catalysis by enolase: an ordered kinetic mechanism for a single substrate enzyme.

Spectroscopic and kinetic methods have been used to explore the roles of divalent metal ions in the enolase-catalyzed dehydration of 2-phosphoglycerate (2-PGA). Enolase requires 2 equiv of metal ion per active site for maximal activity. Previous crystallographic studies [Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) Biochemistry 35, 4349-4358] showed that both magnesium ions coordinated to the carboxylate group of the substrate/product-a scheme consistent with metal ion assistance in formation of the enolate intermediate. Electron paramagnetic resonance (EPR) data with 17O-labeled forms of phosphoenolpyruvate show that Mn(2+), bound at the lower affinity site, coordinates to one carboxylate oxygen and one phosphate oxygen of the substrate. These observations are fully consistent with the crystallographic data. Plots of activity versus log [metal ion] are bell-shaped, and the inhibitory phases of the profiles have been previously attributed to binding of metal ions at ancillary sites on the enzyme. However, the activation profiles and measurements of 2H kinetic isotope effects support an ordered kinetic mechanism wherein binding of 2-PGA precedes binding of the second metal ion, and release of the second metal ion occurs prior to departure of phosphoenolpyruvate. High concentrations of metal ion lead to inhibition in the ordered mechanism by interfering with product release. The 2H kinetic isotope effect is diminished in the inhibitory phases of the metal ion activation profiles in a manner that is consistent with the predominantly ordered mechanism. Zn(2+) gives lower maximal activity than Mg(2+), apparently due to slow release of Zn(2+) from the product complex. Addition of imidazole increases the maximal rate apparently by accelerating the release of Zn(2+) from the enzyme.