Role Of Metal Ions In Biochemistr

Role Of admixturelic element Ions In BiochemistrA coat is a chemical element that is a heartfelt conductor of two electricity and heat and forms cations and ionic bonds with non- coats. In chemical science, a admixture (from classical mtallon, mine) is an element, compound, or alloy char turningerized by high electrical conductivity. In a coat, jots readily lose electrons to form positive ions (cations). Those ions be surrounded by delocalized electrons, which be responsible for the conductivity. The solid thus resurrectd is held by in dynamic fundamental interactions between the ions and the electron blotch, which ar called surfacelic bonds.2 coat ions frivol inhering posts in ab off one 3rd ofenzymes . These ions cig bet modify electron flow I a subst tell or enzyme, thus exerciseively arrestling an enzyme- catalyzed reply. They lavatory serve to take for and orient subst rove with deference to functional bases in the active rate, and they domici liate picture a state of affairs for redox activity if the coat has several(prenominal)(prenominal) valence states. Without the appropriate surface ion, a biochemical reaction catalyzed by a event admixtureloenzyme would progress very slowly, if at all.The enzyme provides an brass of sidechain functional groups having an appropriate surfaced wad with the preferred groups on enzyme side imprisonment needed to keep the necessary admixture ion. The optimal total of more than(prenominal)(prenominal) attach groups is elect for the particular surface ion, together with the appropriate hydrophobic or hydrophilic pur harpu in the spinal column site. Metal ions may be abut by main-chain amino and carbonyl groups, but detail salad dressing is achieved by the amino acid side chains, particularly the treat groups of aspartic and glutamic acid, and the ring due north fragment of histidine. Other side chains that bind surfaces ions include tryptophan (ring nitrogen) , cysteine (thiol), methionine (thioether), serine, threonine, tyrosine (hydroxyl groups), and asparagine and glutamine (carbonyl groups, less(prenominal) practically amino group .No set of general rules exists that describes how a given metallic element ion ordain be down in an enzyme . Now that many a(prenominal) vitreous silica structures of proteins be being canvass by X-ray diffraction, culture on the ski bandaging of metal ions in the active sites of enzymes is available and should provide clues to the mechanism of action of the enzyme.The examples of catechol methyltransferase andmandelate racemase impart be discussed later in this article.The spend a penny described here includes results fromexaminations of the crystal structures in the CambridgeStructural Database and the Protein Databank . Astudy of binding, however, withal involves an analysis ofthe active consequences of changing the way thebinding occurs, so that the closely stable binding figure of speec h fora given group of ligands advise be deduced. We haveapproached this using ab initio molecular orbital and density functional calculations . In this way weobtain both the binding geometry of ligands and theenergetic consequences of changing this binding mode.Properties of metal ionsMetal ions ar generally positively charged and act as electrophiles, seeking the possibility of sharing electron pairs with early(a)(a) atoms so that a bond or charge-charge interaction can be make. They behave rather like henry ions (the poor mans metal). Metal ions, however, frequently have positive charges greater than one,and have a pear-shaped ionic volume so that they can accommodate many ligands or so them at the same(p) time. In addition, metal ion concentproportionns can be high atneutral pH values, man henry ion concentrations ar, by the definition of pH, low at these values. Ligands atomic number 18 the atoms or groups of atoms that are bonded to the metal ion, generally in an motionless trend. They are usually neutral or negatively charged and they give electron density to the metal ion.Thecoordination number of a metal ion, that is, the number of ligand atoms bound to it, is viewed in hurt of concentric scene of actions the familiar sphere take uping those atoms in contact with the metal ion, the aid sphere containing those in contact with the inner sphere ligand atoms. The number of atoms in these spheres will depend on the size of the metal ion and the sizes of the ligand atoms. For example, sodium is elfiner than potassium, and atomic number 16 is larger than group O. Measurements of metal ion-liganddistances in crystal structures led to the idea of atomic and ionic radii 9-11 anion radii can in any case be derived from the minimum anion-anion distances in crystal structures. The radius ratio, a opinion introduced by Goldschmidt 11, is the ratio of the radius of the cation to that of the anion and is generally less than 1.0 Tetrahedr al structures have a radius ratio between 0.225 and 0.414, while octahedral structures have a ratio between 0.414 and 0.645. For example, the radius of Mg2+ is 0.65 D, while that of O2- is 1.40 D and their radius ratio is 0.464 the packing material is octahedral.The charge distribution in the active site of an enzyme is designed to stabilise the transition state of the catalyzed reaction relative to that of the substrate. In enzyme-catalyzed reactions it is essential that the reactants be brought together with the typeset spatial orientation, other(a)wise the chance of the reaction pickings place is diminished and the reaction rate will be in addition low.The electrostatic environment in the active site is a major factor that serves to guide the substrate to the binding site in the correct orientation. Metal ions can assist in this emergence, frequently binding groups in a stereochemically rigid manner, in that locationby helping to control the action of the enzyme. Thus, a n enzyme will bind its substrate in such a manner that immobilizing and alignment, ready formation of the transition state of the reaction to be catalyzed,and and so easy release of the product will result metal ions often help in accomplishing this process.Each metal ion has its own chemistry. An example of the differing reactivities of metal cations is provided by their tycoon to bind or lose water molecules. The transfer of coordinated water with bulk solvent by versatile cations has been categorized into quatern groups those for which the exchange rate is greater than 108 per second including alkali and basic earth metal ions(except beryllium and milligram), together with Cr3+,Cu2+, Cd2+, and Hg2+. Intermediate rate constants (from 104 to 108 per second) are found for Mg2+ and somewhat of the divalent first-row transition metal ions. Those with slow rate constants (from 1 to 104 per second) include Be2+ and certain trivalent first-row transition metal ions. The vacant group with rate from 10-6 to 10-2 per second containsCr3+, Co3+, Rh3+, Ir3+, and Pt2+. superstar of the factors involved in rates of exchange is the charge-to-radius Ratio if this ratio is high the exchange rate is low.An grievous reaction catalyzed by metal ions inenzymes is the ionization of water to give a hydrated hydrogen ion and a hydroxyl anion. sign studies of this process will be discussed here as they are relevant to the action of a metal ion in providing a hydroxyl group and a hydrogen ion for drug abuse in an enzymatic reaction.Polarizing Potential of Various IonsAtoms or groups of atoms are considered polarizable if, when they are placed in an electric field, a charge insularism occurs and a dipole is acquired. This deformability or polarizability is rhythmd by the ratio of the induce dipole to the applied field. Those atoms that hold on less firmly to their electrons are termed more than polarizable. It is found that if twain ions have the same inert botch str ucture (potassium and chloride, for example), the negatively charged anion is more polarizable than the positively charged cation, which holds on to its electrons more tightly. The word hard has been introduced to indicate a low polarizability so that the electron cloud is difficult to deform (like a hard sphere). By contrast indulgent kernel high polarizability so that the electron cloud is readily deformed . A hard acid or metal cation holds tightly to its electrons and thitherfore its electron cloud is not readily garble its unshared valence electrons are not easily excited. Soft (polarizable) metal cations contain electrons that are not so tightly held and in that locationfore are easily distorted or removed.A hard acid prefers tocombine with a hard base, while a soft acid prefers to bind with a soft base by partially forming covalent bonds .The type of binding is related to the highest occupied molecular orbital (HOMO) of the electron-pair donor (a lewis base, the ligand) a nd the lowest inert molecular orbital (LUMO) of the electron-pair acceptor (a Lewis acid, the metal ion). If these have equivalent energies, then electron transfer will give a covalent (soft) interaction, whereas the energy deflexion is large, electron transfer does not readily take place and the interaction is mainly electrostatic (hard-hard).Hardcations include the alkali and alcalescent earth metal ions while soft metal ions include Cu 2+, Hg2 2+, Hg2+, Pd2+. Inbiological systems, hard ligands generally contain oxygen while soft ligands contain sulfur. Hard acids tend to bind hard bases by ionic forces, while soft acids bind soft bases by partially forming covalent bonds. These hard-soft categorizations are a help in understanding the relative binding preferences of conglomerate cations. Most metal ions of biological significance are hard or intermediate between hard and soft. Most soft metal ions and soft ligands are poisonous and they interact with other soft species in th e body. For Pb2+ the harder ligands are found in hemi rateed structures and the softer ligands in holodirected manifoldes.Nature has devised many enzyme systems in which a metal ion interacts with the oxygen of a water molecule.If a water molecule can be dissociated into a hydrogen ion and a hydroxyl group, the latter can serve as a nucleophile in chemical and biochemical reactions.Nature has chosen activation of a water molecule as a means to obtain such a nucleophile in situation so that a chemical reaction can occur in a stereochemically controlled manner in the active site of the enzyme. The questions we ask are as follows 1) how does personality ensure that the peculiar(prenominal) water molecule will be aroused2) how does temper compensate for the lower water activation power of some cations over others (since a large-minded variety of metal ions may not be available in the particular active site and the enzyme has to do the best it can with what is available) and 3) how does spirit ensure that the required reaction occurs.Ab initio molecular orbital and density functional calculations have been carried out to measure the extent to which a series of metal cations can, on binding with water, understanding it to be dissociated into its component hydrogen ions (subsequently hydrated in solution) and hydroxyl ions. Initial data indicate that the charge of the metal ion plays a evidentiary role in modifying the pKa of water. The binding enthalpies of a wide variety of metal ion monohydrates, MH2O2+ , have been published 21 but their deprotonation enthalpies are still under investigation.Geometry of Metal-Ion Binding to structural GroupsThe geometries of metal ion- treat interactions have been studied in order to determine the quest1)which lone pair of an oxygen atom in a carboxylate group, syn or anti, is preferred for metal cation binding2) does the metal ion lie in the plane of the carboxylgroup and3) under what conditions do metal ions shareboth oxygen atoms of the carboxylate group equally? We found that cations generally lie in the plane of the carboxylate group . The exceptions to this mainly include the alkali metal cations and some alkaline earth cations these metals ionize readily and form unassailable bases so it is not surprising that they have less specific binding modes. When the distance of the metal cation to the carboxylate oxygen atoms is on the order of 2.3-2.6 D, the metal ion tends to share both oxygen atoms equally.Otherwise one oxygen atom of the carboxylate group is bound to the metal ion and the other is not. Calcium ions often form bidentate interactions, while it is less common for the microscopicer milligram ions. Imidazole groups in histidyl side chains of proteins bind metal ions in a variety of enzymes. One imidazole can, by virtue of its two nitrogen atoms, bind one or two metal ions, depending on its ionization state and the suitabilities of the metal ion. The bases in desoxyribonucleic acid can in like manner bind metal ions. We have analyzed hydrogen bonding to and from nitrogen atoms in nitrogen-containing heterocycles for crystal structures in the CambridgeStructural Database. It was found that for hydrogen bonding, a slight out-of-plane deviation of the binding atom often occurs. Metal ions bind more smack in the plane of the imidazole group. The energetic court of such deviations were analyzed by ab initio molecular orbital calculations. In an investigation of protein crystal structures in the Protein Databank it was found that the binding of metal ions to histidine in proteins is more rigid and the location of the metal ion is more directional.Thus, if an enzyme needs to control the location and orientation of a carboxylate or imidazole group, it can accomplish this better with a metal ion than by hydrogen bonding.Metal ions in proteins are often involved in structural motivations. When a metalloenzyme carries out its catalytic function it uses one of a few executable three-dimensional arrangements of functional groups around the metal ion to ensure the specificity of the required biochemical reaction. Thus, if such catalytic metal-binding motifs can be determine and categorized, then inchoate reactivities of enzymes could be inferred from their three-dimensional structures. Such a categorization, however, requires an understanding of the underlying chemistry of any metal ion in the active site.One motif identified in the crystal structure of cobalt(II) formate consists of a carboxyl group in which one oxygen atom is bound to the metal ion and the other is bound to metal-bound water, to give a cyclic structure.This motif has been found in many metalloenzyme crystal structure , such as D-xylose isomerase.The roles of these motifs are of interest. The metal ion-hydrated-carboxylate motif (I) is planar and commonly found. It does not, however, affect the ability of the metal ion (in studies of Mg2+ complexes) to ionize water. On the othe r hand, for magnesium ions (which generally have a rigid octahedral arrangement of binding groups) it utilizes 2 of the 6 coordination locatings and therefore serves to orient the arrangement of ligands, an put together we have labeled coordination clamping. Motif (II) is also found in several crystal structures such as that of the -subunit of integrin CR3 . It seems to help bind subunits together.A third motif (III) is found in D-xylose isomerase and involves two metal ions with several carboxylate ligands and a histidine ligand . The metal site that binds only oxygen atoms can bind substrate in place of the two water molecules and orient the substrate. The second metal ion site (with histidine as one ligand) then positions a metal ion-bound water molecule to attack the substrate.Roles of Metal Ions in Enzyme ActionThe crystal structure of mandelate racemase with bound p-iodomandelate provides a useful example of the importance of a metal ion in a reaction . The enzyme binds a magnesium ion by means of three carboxyl groups. The substrate mandelate has send awayd water from the magnesium coordination sphere and binds by means of its carboxylate group and an a-hydroxy group.The magnesium ion will lie in the plane of the carboxyl group, as shown by our studies of metal ion-carboxylate interactions . The magnesium holds the substrate firmly in place so that the catalytic precis and addition of a hydrogen atom by His 297 or Lys 166 is exactly effected . The magnesium probably also aids this activity by affecting the electronic flow in the carboxylate and hydroxyl groups by mild polarization. We have found that metal ion coordination is better than a hydrogen bond in aligning a functional group there is considerable flexibility in a hydrogen bond as we found for imidazoles . In the reaction catalyzed by the enzyme mandelate racemase the magnesium ion binds substrate . A Histidine (His 297) and Lysine (Lys 168) are positioned to abstract a hydrogen ion from the substrate and, if it is added again from the other side, racemization occurs. Hydrogen bonding to a carboxylate group of the substrate helps to stabilize an enolate intermediate in the reaction.In catechol O-methyltransferase , a methyl group is transferred from the sulfur of Sadenosy methionine to catechol. The magnesium ion is oriented by a motif of type I and it binds substrate in such an orientation that a hydroxyl group is near the S-CH3 group, and the other hydroxyl group is held in place by a carboxylate group. on that point are many other examples of two-metal ion active sites, such as hemerythrin, alkaline phosphatase and superoxide dismutases (which have been well documented). These studies of the geometries and energetics of metal-ion ligand b inding can therefore aid in our understanding of metalloenzyme functionMetals in the ribonucleic acid woridBy combining our limited knowledge of metal-ion-binding to contemporary RNAs and our more extensive knowledge of metal -ion-binding to proteins, it is possible to speculate on the role of metal ions in prebiotic molecular evolution. It seems clear that specifically bound metal ions coevolved with RNA molecules. Many of the mononuclear sites in Table 5 are formed with, or can be engineered into, small RNA fragments. Since such sites are highly hydrated and contain limited direct contact with the RNA, the spy affinities are only moderate, in the 1-1000 M range.These sites are also pass judgment to show limited specificity, predominantly dictated by the chemical nature of the ligands. Furthermore, in these examples, the RNA structures themselves are promising to be quite tensile and can accommodate a variety of metal ions with only squirt distortions to the overall RNA fold. These minimalist sites are sufficient to stabilize the lowly and tertiary structures observed in these motifs.The metal ion sites generated on small RNAs appear to be capable of facilitating a variety of different types of ch emistry. Activities range from the transesterification and hydrolytic reactions of small ribozymes (Pyle 1996 Sigurdsson et al. 1998) to the more exotic porphyrin metalation (Conn et al. 1996) and Diels-Alder condensation reactions (Tarasow et al. 1997) catalyzed by aptamers produced from in vitro selection experiments.These small RNAs have only limited amounts of structure and therefore are likely to position the catalytic metal ions by only a few points of contact. The relatively modest rate enhancements supported by catalytic RNAs such as these probably job the types of species that first evolved from random polymerization events. truly active metal ions might have assisted in this process but would have increased the danger of side reactions that would accidentally modify the catalyst.A striking difference between most RNA metal-binding sites studied thus far and those seen in proteins is the degree of hydration. Both structural and catalytic metal-ion-binding sites in protei ns are predominantly dehydrated (Lippard and Berg 1995). Water molecules on occasion appear in the coordination spheres of these metal ions, but in these cases, they are often believed either to be displaced by the substrate when it enters the active site or to take part in the catalytic mechanism of the enzyme. Such protein sites also bind their metal ions very much more tightly than the RNA systems. In fact, tight binding is a requirement for dehydrated sites, since there is a characteristic energy (Hhyd) associated with the hydration of any ion. The net binding energy upon coordination of the ion moldiness account for the energetic cost of dehydration.The question arises, Why are such dehydrated sites not observed in RNAs? One possibility is that metal-binding sites in RNAs are intrinsically different from those in proteins. RNA has a much more limited set of ligands to use in generating a specific metal-binding pocket. Amino acid side chains containing thiols and thioethers a re well desirable to binding a variety of softer metals. In addition, the carboxylate side chains provide anionic ligands with great versatility in their potential modes of coordination. They can act as either terminal or bridging ligands and bind in either monodentate or bidentate geometries. The nucleotides, on the other hand, are much larger and more rigid than the corresponding amino acids. The anionic ligand in RNA, the nonbridging inorganic phosphate oxygen, is an integral component of the backbone and therefore is more limited in its conformational freedom than the aspartate and glutamate carboxylate groups. The heterocyclic ring nitrogens and the keto oxygens from the bases are held in rigidly planar orientations by the aromatic rings. This geometric constraint severely limits the ability of an RNA to compact encompass a metal ion and provide more than facial coordination and therefore complete dehydration. It also explains why the most specific metal-binding sites are not in the Watson-Crick base-paired regions of the structure where the conformation is too constrained. Instead, metalion- binding sites are clustered in regions of extensive distortion from the A-form RNA helices.There is also the question of the flexure of RNAs relative to that of proteins. It is possible that in RNAs there is insufficient energy in the folding and metal-binding process to completely displace the waters of hydration around a metal ion. It has been suggested that in contemporary RNAs, modified nucleotides might be present to assist in metal ion binding (Agris 1996). A more straightforward possibility, however, is that most RNAs studied to date are structurally too simple. In these RNAs, most residues involved in metal ion binding are solvent-exposed. Thus, the RNAs have no real inside comparable with(predicate) to the hydrophobic load of a protein. The largest RNA crystallographically characterized to date is the P4-P6 domain. On the foothold of that structure, i t was proposed that an ionic core may substitute in RNA folding for the hydrophobic core of proteins such that the 3 structure assembles around a fixed number of discrete metal-binding sites (Cate et al. 1997). Even in this structure, however, the most bury of the metal-binding sites are importantly hydrated. It could be that all metal-ion-binding sites in RNA are at least partially hydrated. One can imagine several advantages to using hydrated ions within the ionic core of a large RNA. Hydrated ions would span larger voids than dehydrated ions and allow looser packing of secondary structure elements. The hydrated ion also can accommodate a wide range of structural interactions through its orientation of the water molecules as compared to direct coordination of metal ions at every site. In addition, the energy associated with deforming the outer-sphere interactions should be significantly less than what would be observed for distorting the innersphere coordination. A consequence of RNAs having a core of hydrated ions is that one might expect this core to be much more dynamic than the hydrophobic core of a protein.In the modernistic protein world, metal cofactors are associated with a variety of reaction types, including electron transfer, redox chemistry, and hydrolysis reactions. Trans esterification and hydrolytic activities, however, are the primary catalytic behaviors observed in ribozymes. Did these other catalytic activities not develop until the dawn of the protein world, or are there unexplored natural catalytic RNAs that are the ancestors of the early redox enzymes? Through the use of in vitro selection experiments, the scope of RNA catalysis has been significantly broadened is or so certainly capable of catalyzing these other classes of reactions, but it is still unclear whether there are naturally occurring examples. Such an enzyme would likely use a metal ion cofactor other than Mg(II), so the search for RNA molecules that naturally use altern ative ions is of significant interest. A recent selection experiment showed that a whizz base change results in an altered metal ion specificity for RNase P (Frank and Pace 1997). It is clear from this result that catalytic RNAs retain the abilityto accommodate to an everchanging environment, using the resources available to evolve and to overcome evolutionary pressures. Were RNAs to have evolved out of an environment devoid of metal ions, they probably would have found a way around the problems of folding and generating reactive functional groups. The primordial dope up and all cellular environments that have evolved subsequently contained a variety of ions, however. stipulation the availability of metal ions, they will certainly play a significant role in the biology of current and future RNAs.Effect of metal ions on the kinetics of tyrosine oxidation by TyrosinaseThe conversion of tyrosine into dopa 3-(3,4-dihydroxyphenyl)alanine is the rate limiting step in the biogenesis o f melanins catalysed by tyrosinase. This hydroxylation reaction is characterized by a lag period, the extent of which depends on various parameters, notably the front of a adequate hydrogen donor such as dopa or tetrahydropterin. We have now found that catalytic amounts of Fe2+ ions have the same effect as dopa in stimulating the tyrosine hydroxylase activity of the enzyme. kinetic experiments showed that the shortening of the induction time depends on the concentration of the added metal and the nature of the buffer system used and is not suppressed by superoxide dismutase, catalase, formate or mannitol. Notably, Fe3+ ions showed only a small delaying effect on tyrosinase activity. Among the other metals which were tested, Zn2+, Co2+, Cd2+ and Ni2+ had no detectable influence, whereas Cu2+ and Mn2+ exhibited a marked inhibitory effect on the kinetics of tyrosine oxidation. These findings are discussed in the light of the commonly judge mechanism of action of tyrosinase. Tyrosin ase (monophenol,dihydroxyphenylalanine oxygen oxidoreductase is a copper-containing enzyme responsible for melanogenesis in plants and animals, which catalyses both hydroxylation of tyrosine to dopa and its subsequent oxidation to dopaquinone (Hearing et al., 1980 Lerch, 1981). The first reaction, which represents the rate-limiting step in melanin biosynthesis (Lerner et al., 1949), is characterized by a lag period that has subsequently been explained in terms of a hysteretic process of the enzyme (Garcia Carmona et al., 1980). The extent of this induction time depends on various parameters including, besides pH and both substrate and enzyme concentration, the front of a suitable hydrogen donor.Kinetic studies carried out on tyrosinases from various sources (Pomerantz, 1966 Pomerantz Murthy, 1974 Hearing Ekel, 1976 Prota et al Abbreviations used dopa, 3-(3,4-dihydroxyphenyl)-alanine SOD, superoxide dismutase. To whom correspondence and reprint requests should be addressed. 1981) have shown that dopa, in very low concentration, is the most effective reducing agent in eliminating the lag period, whereas other catechols, such as dopamine, adrenaline and noradrenaline, behave similarly to ascorbate and NADH and NADPH in only shortening it, even at high concentration. Tetrahydropterin, a well-known specific cofactor of other aromatic hydroxylases (Lerner et al., 1977 Marota Shiman, 1984), is also effective in stimulating tyrosinase activity, although to a lesser extent than dopa. At present, no other organic or inorganic substances have been reported to shorten or lengthen the lag period of tyrosine oxidation. Although metal ions are known to play a role in many biological processes, little attention has been directed to their possible involvement in melanogenesis, particularly in the early enzymic stages .As a part of our continuing studies on the chemistry of melanin pigmentation (Prota, 1980 Sealey et al., 1982 Palumbo et al., 1983), we report the results o f a survey on the effect of metal ions on the activity of purified burnt sienna tyrosinase, readily available in large amounts from the ink of the cephalopod genus Sepia officinalis thermostability of amalyseThree Metal Ions Participate in the Reaction Catalyzed by T5 cockle Endonuclease*-Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the fast hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are wide proposed to catalyze phosphate diester hydrolysis using a two-metal-ion mechanism. Yet, analyses of beat up endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, act controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of4A appears incompatible with this mecha nism. To clarify the roles compete by FEN metal ions, we report here a flesh out evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2_ or Mn2_). The apparent substrate affiliation constant is maximized by binding two magnesium ions. This may reflect a metal dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN in general uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.Key cellular processes such as DNA repl ication, DNA repair, RNA processing, and RNA degradation require the rapid hydrolysis of the phosphate diester linkages of nucleic acids. The uncatalyzed hydrolysis of phosphate diesters under biological conditions is an extremely slow process with an estimated half-life of 30 million years at 25 C (1). Protein nucleases and RNA enzymes produce rate enhancements of 1015-1017 to allow this reaction to proceed on a biologically useful time scale.Most enzymes catalyzing phosphate diester bond hydrolysis have a requirement for divalent metal ions. Based largely upon crystallographic observations, most metallonucleases are proposed to catalyze reactions using a two-metal-ion mechanism (Fig. 1a) analogous to that suggested for the phosphate monoesterase alkaline phosphatase (2, 3), although this view is not universally accepted. Three recent reviews present tell apart views on the roles of metal ions in protein nuclease andRNA enzyme reactions and illustrate this controversy (4-6). One f amily of metallonucleases over which there has been considerable mechanistic debate are the flap endonucleases (FENs)3 (7-12), which are present in all domains of life and play a key role in DNA replication and repair. Unlike most metallonucleases, which typically possess a cluster of three or four active site carboxylates, the FEN active site is constructed from seven or eight acidic residues located in similar positions in FENs from a range of organisms (Fig. 1b, see also supplemental Fig. S1) (7, 9, 10, 13-16). Several FEN roentgen ray structures also contain two active site carboxylate-liganded divalent metal ions, designated as metals 1 and 2 (9, 13-15). The position of metal 1 is similar in all cases, but the metal 2 location varies. In all but on