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PubMed1995

Residues affecting the catalysis and inhibition of rat lens aldose reductase.

Aldose reductase (AR), the first enzyme of the polyol pathway, has been implicated in diabetic complications. Results of recent clinical studies have shown that compounds that inhibit aldose reductase (ARIs) and block the flux of glucose through the polyol pathway have provided benefit to diabetic neuropathic patients. Since many ARIs show broad substrate specificity, emphasis on the structure-function properties of the AR enzyme will help in the refinement and design of future inhibitors. To this end, catalysis and inhibition of rat lens aldose reductase was examined following site-directed mutagenesis. Replacement of tyrosine 48 with phenylalanine (Y48F) resulted in an enzyme form with less than 0.25% activity with DL-glyceraldehyde and no detectable activity with p-nitrobenzaldehyde or xylose, although circular dichroism spectra and NADPH binding affinity were similar to wild-type AR. Mutation of histidine 110 to glutamine (H110Q) also resulted in a less active protein with an approximate 3-fold decrease in kcat for the reduction of DL-glyceraldehyde; slight or no activity was measured with other substrates and an increase of 195-fold over wild type was observed in the Km for glyceraldehyde. H110Q was less sensitive to inhibition by aldose reductase inhibitors. The most dramatic change was seen with imeristat, which showed an 1800-fold increase in IC50. Mutation of cysteine 298 to serine (C298S) affected enzyme function by increasing kcat 2- to 4-fold and increasing Km 15- to 48-fold, with DL-glyceraldehyde, p-nitrobenzaldehyde or xylose as substrates. As a result kcat/Km, catalytic efficiency, dropped to approx. 10% of control. Inhibition of C298S was not noticeably different from wild type. Substitution of histidine 187 or 200 with glutamine (H187Q, H200Q) had little effect on AR catalysis or inhibition. Based on structural and mutagenesis studies of human AR and the conservation of amino acids between human and rat, these data would indicate that Y48, H110, and C298 are important residues in the active site of rat AR and that Y48 is most likely the proton donor during substrate reduction by rat lens aldose reductase. In addition, these studies indicate that mutagenesis of H110 also affects aldose reductase inhibition.

Aldehyde Reductase↗
PubMed1994

Isotope effects on the mechanism of calcineurin catalysis: kinetic solvent isotope and isotope exchange studies.

The reaction scheme of calcineurin was examined with kinetic and physical approaches. Proton inventory studies of the calcineurin-catalyzed hydrolysis of para-nitrophenyl phosphate were done to probe the role of proton transfer in the mechanism. Control experiments determined that the solvent did not cause the irreversible inactivation of the enzyme and had no effect on the dependence on metal ion or calmodulin. A solvent isotope effect was observed on the Vmax/Km term, but not the Vmax term. The isotope effect was modest with a value of 1.35. Proton inventory data could be fit by multiple parameter sets. The parameter sets yielded fractionation factors of 0.73 for a one-proton transfer or 0.85 for a two-proton transfer. These values compare to the value of 0.69 for reactions involving a water molecule or hydroxide coordinated to metal ion. A chemical mechanism consistent with the proton inventory data and other information about calcineurin catalysis is presented. The simplest model for catalysis involves a single proton transfer from water coordinated to metal that is reasoned to occur during association of the substrate with calcineurin. Questions about the reaction intermediate were also addressed. Attempts to monitor a phosphate-water exchange reaction with 31P nuclear magnetic resonance spectroscopy were unsuccessful. Failure to observe an exchange reaction suggests that no phosphoryl enzyme is formed during the progress of the reaction. Together these data are explained by a model in which cleavage of the phosphate ester bond is catalyzed by a water (hydroxide) molecule coordinated to a divalent metal ion without the formation of a covalent intermediate.

Animals↗
PubMed1995

Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.

Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases) EcoRI and EcoRV, because experimental evidence exists that the phosphate group 3' to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (BglII, BstI, BstYI, Cfr10I and MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BamHI, Bsp143I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, DpnII, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MflI, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistance in catalysis.

Base Sequence↗
PubMed2005

Probing the role of tightly bound phosphoenolpyruvate in Escherichia coli 3-deoxy-d-manno-octulosonate 8-phosphate synthase catalysis using quantitative time-resolved electrospray ionization mass spectrometry in the millisecond time range.

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. The enzyme is often isolated with varying amounts of tightly bound PEP substrate. To better understand the role of tightly bound PEP in E. coli KDO8P synthase catalysis, a combination of transient kinetic methodologies including rapid chemical quench and mass spectrometry techniques such as time-resolved electrospray ionization mass spectrometry (ESI-TOF MS) were used to study the enzyme purified both in the PEP-bound state and in the unbound state. Pre-steady state burst and single-turnover experiments using radiolabeled [1-(14)C] and [(32)P]A5P revealed significant kinetic differences between these enzyme preparations. The active sites concentrations for the bound and unbound states of the enzyme were almost the same (approximately 100%) and the product release for both states of the enzyme was rate limiting. However, the rate constant of product formation for the PEP-bound enzyme (125 s(-1)) was higher than that of the unbound enzyme (46 s(-1)). This was further confirmed by single-turnover experiments using radiolabeled [(32)P]A5P. Interestingly, when PEP was removed from the PEP-bound enzyme and external PEP was added before the kinetic experiments, both the pre-steady state burst and the single-turnover kinetic parameters were similar to those of the enzyme purified in the unbound state. The rate constants of product formation were determined as 44 s(-1) (burst experiment) and 48 s(-1) (single-turnover experiment). The reaction kinetics of the E. coli KDO8P synthase was also followed by time-resolved ESI mass spectrometry. To validate the suitability of this technique for conducting enzyme kinetics, the standard reaction of p-nitrophenyl acetate hydrolysis by chymotrypsin was analyzed by stopped-flow and time-resolved ESI-TOF MS. The rate constant of p-nitrophenol formation followed by stopped-flow spectrophotometry matched perfectly the rate constant of acetyl-chymotrypsin intermediate formation followed by time-resolved ESI-TOF MS (0.1 s(-1)). The catalytic properties of the PEP-bound and unbound states of the E. coli KDO8P synthase were then studied on a millisecond time scale. The changes in the intensity of E*PEP, E*KDO8P, and E*intermediate complexes as a function of time were quantified and the reaction kinetics were modeled using KinTekSim simulation software. An analysis of the reaction kinetics established the kinetic competence of the intermediate based upon the rate constants for substrate decay and product formation. The ability of time-resolved ESI-TOF MS to detect and monitor the kinetics for the reaction intermediate constitutes a significant advantage over the traditional rapid chemical quench technique. For all three states of the enzyme (PEP-bound, unbound, and PEP removed from the PEP-bound state) the rate constants obtained by time-resolved ESI-TOF MS matched the pre-steady state rates determined by rapid chemical quench. A comparison of reaction time courses for each state of the enzyme revealed that, in the case of PEP-bound enzyme, the enzymatic reaction reached completion faster than that for the unbound state. In summary, these studies led to the conclusion that bound PEP has an important role in catalysis, maintaining the enzyme in a conformational state optimal for catalytic activity, and established the kinetic competence of the reaction intermediate. This technique has broad applicability for the kinetic analysis of any enzyme system where the substrates, products, or intermediates are eluding the common detection techniques or as a method alternative to the widely used radioactivity assays.

Aldehyde-Lyases↗
PubMed2004

The impact of metal catalysis on protein tyrosine nitration by peroxynitrite.

In a series of heme and non-heme proteins the nitration of tyrosine residues was assessed by complete pronase digestion and subsequent HPLC-based separation of 3-nitrotyrosine. Bolus addition of peroxynitrite caused comparable nitration levels in all tested proteins. Nitration mainly depended on the total amount of tyrosine residues as well as on surface exposition. In contrast, when superoxide and nitrogen monoxide (NO) were generated at equal rates to yield low steady-state concentrations of peroxynitrite, metal catalysis seemed to play a dominant role in determining the sensitivity and selectivity of peroxynitrite-mediated tyrosine nitration in proteins. Especially, the heme-thiolate containing proteins cytochrome P450(BM-3) (wild type and F87Y variant) and prostacyclin synthase were nitrated with high efficacy. Nitration by co-generated NO/O(2)(-) was inhibited in the presence of superoxide dismutase. The NO source alone only yielded background nitration levels. Upon changing the NO/O(2)(-) ratio to an excess of NO, a decrease in nitration in agreement with trapping of peroxynitrite and derived radicals by NO was observed. These results clearly identify peroxynitrite as the nitrating species even at low steady-state concentrations and demonstrate that metal catalysis plays an important role in nitration of protein-bound tyrosine.

Catalysis↗
PubMed2005

Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases.

Flavoprotein monooxygenases are involved in a wide variety of biological processes including drug detoxification, biodegradation of aromatic compounds in the environment, biosynthesis of antibiotics and siderophores, and many others. The reactions use NAD(P)H and O2 as co-substrates and insert one atom of oxygen into the substrate. The flavin-dependent monooxygenases utilize a general cycle in which NAD(P)H reduces the flavin, and the reduced flavin reacts with O2 to form a C4a-(hydro)peroxyflavin intermediate, which is the oxygenating agent. This complicated catalytic process has diverse requirements that are difficult to be satisfied by a single site. Two general strategies have evolved to satisfy these requirements. para-Hydroxybenzoate hydroxylase, the paradigm for the single-component flavoprotein monooxygenases, is one of the most thoroughly studied of all enzymes. This enzyme undergoes significant protein and flavin dynamics during catalysis. There is an open conformation that gives access of substrate and product to solvent, and a closed or in conformation for the reaction with oxygen and the hydroxylation to occur. This closed form prevents solvent from destabilizing the hydroperoxyflavin intermediate. Finally, there is an out conformation achieved by movement of the isoalloxazine toward the solvent, which exposes its N5 for hydride delivery from NAD(P)H. The protein coordinates these dynamic events during catalysis. The second strategy uses a reductase to catalyze the reduction of the flavin and an oxygenase that uses the reduced flavin as a substrate to react with oxygen and hydroxylate the organic substrate. These two-component systems must be able to transfer reduced flavin from the reductase to the oxygenase and stabilize a C4a-peroxyflavin until a substrate binds to be hydroxylated, all before flavin oxidation and release of H2O2. Again, protein dynamics are important for catalytic success.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2004

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: relevance of arginine 70 for catalysis.

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg70, whose guanidinium group is close to the PEP carboxyl group. Arg70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg70Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.

Adenosine Diphosphate↗
PubMed2004

On the importance of being zwitterionic: enzymatic catalysis of decarboxylation and deprotonation of cationic carbon.

Carbanion ylides are strongly stabilized by electrostatic interactions between opposing charges at neighboring atoms and this stabilizing electrostatic interaction increases with decreasing dielectric constant of the medium through which the charges interact. Consequently, there is a large increase in the thermodynamic driving force, with decreasing dielectric constant of the reaction medium, for deprotonation of cationic carbon acids and decarboxylation to form related ylides. This favors catalysis of the formation of unstable ylides at enzyme active sites of low dielectric constant. A brief survey of enzymes that catalyze deprotonation of cationic carbon acids and related decarboxylation reactions shows catalysis generally occurs for substrates that are bound in a deep pocket on the protein, with an apparent dielectric constant that is much lower than for the solvent water. In several cases, proton transfer is to a catalytic residue that is relatively weakly solvated in water. We suggest that there is a strong advantage for evolution of protein catalysts that utilize weakly solvated basic side chains which are relatively easily buried in nonpolar active sites that are favorable for zwitterion formation.

Amino Acids↗
PubMed2006

Nucleobase catalysis in ribozyme mechanism.

RNA performs a wide range of functions in biology including catalysis of chemical reactions. A major goal in the field of ribozyme chemical biology is to understand these functions in molecular terms. There is increasing evidence that ribozymes can use their nucleobases directly in chemical catalysis in a variety of ways. These include hydrogen bonding to the transition state, stabilizing charge development, and transferring protons as general acid-base catalysts. This article highlights recent kinetic, structural, single molecule, and synthetic approaches that have been used to probe the roles of ribozyme nucleobases in phosphodiester bond cleavage.

Catalysis↗
PubMed2005

Functional characterization of medaka CYP3A38 and CYP3A40: kinetics and catalysis by expression in a recombinant baculovirus system.

Phylogenic analysis of the teleost genomic lineages has demonstrated the precedent for multiple genome duplications. Among many of the genes duplicated, cytochrome P450 genes have undergone independent diversification, which can be traced to a single ancestral gene. In teleosts, cytochrome P450s, from all major families, have been identified. Among these, the CYP3A family has been cloned in several teleost species and demonstrated to contain multiple paralogs differing in gene expression patterns and tissue distribution. Herein we characterized the catalytic and kinetic activities of two medaka CYP3A paralogs (CYP3A38 and CYP3A40) with benzyloxyresorufin (BFC), a fluorescent 3A-selective substrate, and testosterone, a known metabolic substrate for CYP3A enzymes. Recombinant CYP3A was produced using the baculovirus expression vector system in Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn5) insect cells and accounted for up to 24% of total cellular protein. Following addition of a heme-albumin conjugate to log phase cells, spectral P450 content reached a maximum of 560 and 2350 pmol/mg microsomal protein for CYP3A38 and CYP3A40, respectively. Incubations containing recombinant CYP3A, human NADPH-cytochrome P-450 oxidoreductase reductase, human cytochrome b5, and a NADPH generation system catalyzed the dealkylation of BFC and hydroxylation of testosterone with a high degree of stereoselectivity. However, efficiencies and specificities were significantly different between the two isoforms. Km and Vmax activities based on BFC-catalysis were 0.116 and 0.363 muM, and 7.95 and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. CYP3A38 preferentially catalyzed testosterone hydroxylation at the 6beta-, 2beta- and 16beta-positions with minor hydroxylation at other positions within the steroid nucleus. Testosterone catalysis with CYP3A40 was limited predominantly to the 6beta- and 2beta-positions. Putative identification of CYP3A substrate recognition sites (SRS) 1-6 indicates that 12 of the 49 amino acid differences between CYP3A38 and CYP3A40 OFRs occur in SRS regions previously known to be associated with steroid hydroxylation. We suggest that differences in kinetics and catalytic activities are a result of amino acid substitutions in SRS regions 1, 3 and 5 within the CYP3A38 and CYP3A40 protein sequence.

Amino Acid Sequence↗
PubMed2005

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis.

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.

Arabidopsis Proteins↗
PubMed2004

Protein motions promote catalysis.

A relationship between molecular dynamics motions of noncatalytic residues and enzyme activity has recently been proposed. We present examples where mutations either near or distal from the active site residues modify internal enzyme motion with resulting modification of catalysis. A better understanding of internal protein motions correlated to catalysis will lead to a greater insight into enzyme function.

Animals↗
PubMed2004

Novel role of tyrosine in catalysis by human AP endonuclease 1.

Apurinic/apyrimidinic endonuclease (AP endo, HAP1) recognizes abasic sites in ds DNA and makes a single nick in the backbone 5' to the abasic site. In this report we examine the roles of three conserved tyrosine residues in close proximity to the active site. We show that Tyr(128) and Tyr(269), which interact upstream and downstream of the abasic site, respectively, are involved in recognition and binding of abasic site-containing double stranded DNA. However, the two residues are not equivalent, as their effects are differentiated by changes in salt concentration. In sharp contrast, Tyr(171) is directly involved in catalysis as well as binding. Y171F, Y171H, and Y171A all show decreased catalytic efficiencies 25,000-50,000-fold from the WT enzyme. Both imidazole and basic pH markedly stimulate the WT enzyme. Imidazole stimulates Tyr(171) mutant enzymes when tyrosine is also present but basic pH eliminates remaining mutant activity. These results underscore the importance of tyrosines in AP endo catalysis. They render the current hypotheses regarding enzyme action unlikely and allow us to consider the possibility that the phenolate of Tyr(171) is the nucleophile that attacks the scissile phosphate.

Amino Acid Substitution↗
PubMed2004

Conformational change in substrate binding, catalysis and product release: an open and shut case?

The role of conformational change in substrate binding, catalysis and product release is reviewed for 11 enzymes, for which crystal structures are available for the apo, substrate- and product-bound states. The extent of global conformational changes is measured, and the movements of the functional regions involved in catalysis and ligand binding are compared to the rest of the structure. We find that most of these enzymes undergo relatively small amounts of conformational change and particularly small changes in catalytic residue geometry, usually less than 1 A. In some enzymes there is significant movement of the binding residues, usually on surface loops.

Catalysis↗
PubMed2004

Leaving group activation by aromatic stacking: an alternative to general acid catalysis.

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.

Acids↗
PubMed2006

Internal bulge and tetraloop of the catalytic domain 5 of a group II intron ribozyme are flexible: implications for catalysis.

RNA molecules have an inherent flexibility that enables recognition of other interacting partners through potential disorder-order transitions, yet studies to quantify such motional dynamics remain few. With an increasing database of three-dimensional structures of biologically important RNA molecules, quantifying such motions becomes important to link structural deformations with function. One such system studied intensely is domain 5 (D5) from the self-splicing group II introns, which is at the heart of its catalytic machinery. We report the dynamics of a 36 nucleotide D5 from the Pylaiella littoralis group II intron in the presence and absence of magnesium ions, and at a range of temperatures (298K-318 K). Using high-resolution NMR experiments of heteronuclear nuclear Overhauser enhancement (NOE), spin-lattice (R(1)), and spin-spin (R(2)) (13)C relaxation rates, we determined the rotational diffusion tensor of D5 using the ROTDIF program modified for RNA dynamic analysis (ROTDIF_RNA). The D5 rotational diffusion tensor has an axial symmetric ratio (D(||)/D(perpendicular)) of 1.7+/-0.3, consistent with an estimated overall rotational correlation time of tau(m)=(2D(||)+4D(perpendicular))(-1) of 6.1(+/-0.3) ns at 298 K and 4.1(+/-0.2) ns at 318 K. The measured relaxation data were analyzed with the reduced spectral density mapping formalism using assumed values of the chemical shift anisotropy of the (13)C spins. Both the relaxation data and the values of the spectral density function reveal that the functional groups in D5 implicated in magnesium ion binding and catalysis (catalytic triad, internal bulge, and tetraloop regions) exhibit thermally induced motion on a wide variety of timescales. Because these motions parallel those observed in the intramolecular stem-loop of the U6 element within the spliceosome, we hypothesize that such extensive dynamic disorder likely facilitates D5 engaging both binding and catalytic regions of the ribozyme, and these may be a conserved feature of the catalytic machinery essential for catalysis.

Anisotropy↗
PubMed2006

Role of each residue in catalysis in the active site of pyrimidine nucleoside phosphorylase from Bacillus subtilis: a hybrid QM/MM study.

Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. We have built a model of a closed active conformation of the three-dimensional structure of PYNP from Bacillus subtilis. Using docking, molecular dynamics, and hybrid quantum-mechanical/molecular-mechanical methods to study the reaction mechanics between PYNP and a substrate, we identified the role of each residue in the active site during the entire catalytic process. The results indicate that the function of His(82), Arg(169), and Lys(188) is to stabilize the uridine in a high-energy conformation by means of electrostatic interactions and that these residues are involved in catalysis. In addition, the function of Asp(162) is likely to activate Lys(188) for phosphorolytic catalysis through polarization effects.

Amino Acids↗
PubMed2006

Making and breaking nucleic acids: two-Mg2+-ion catalysis and substrate specificity.

DNA and a large proportion of RNA are antiparallel duplexes composed of an unvarying phosphosugar backbone surrounding uniformly stacked and highly similar base pairs. How do the myriad of enzymes (including ribozymes) that perform catalysis on nucleic acids achieve exquisite structure or sequence specificity? In all DNA and RNA polymerases and many nucleases and transposases, two Mg2+ ions are jointly coordinated by the nucleic acid substrate and catalytic residues of the enzyme. Based on the exquisite sensitivity of Mg2+ ions to the ligand geometry and electrostatic environment, we propose that two-metal-ion catalysis greatly enhances substrate recognition and catalytic specificity.

Catalysis↗