Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Glycerolipid biosynthesis in rat adipose tissue. I. Properties and distribution of glycerophosphate acyltransferase and effect of divalent cations on neutral lipid formation

A sensitive radioactive assay of acyl CoA:sn-glycerol-3-phosphate-O-acyltransferase (EC 2.3.1.15) was developed to study the properties and subcellular distribution of this enzyme in rat epididymal adipose tissue. The esterification of sn-glycerol-3-phosphate was measured in the presence of palmitoyl CoA or palmitate, ATP, CoA, and Mg(2+) at pH 7.5. The presence of glycerophosphate acyltransferase was detected in both mitochondria and microsomes. The product of this reaction was identified as phosphatidate by thin-layer chromatography and dual isotope incorporation studies. Several divalent cations reduced the activity of this enzyme. Although Mg(2+) was not required for the activity of glycerophosphate acyltransferase, its addition to the incubation mixture resulted in an increased formation of neutral lipids at the expense of phosphatidate. This result is explained by an activation of microsomal phosphatidate phosphatase (EC 3.1.3.4). The effect of Mg(2+) was completely abolished by Ni(2+), Co(2+), Mn(2+), and Zn(2+). These studies suggest that the balance between Mg(2+) and several other divalent ions may be important in the regulation of neutral lipid synthesis in adipose tissue.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Substrate and metal ion promiscuity in mannosylglycerate synthase

The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.     

Effect of metallic cations on the viability of phenol-treated Escherchia coli

Five metallic cations (Fe(3+), Cr(3+), Ca(2+), Mg(2+), Mn(2+); concentration range, 1.85 x 10(-4) to 37 x 10(-4)m) were incorporated individually as chlorides into nutrient broth and agar media used for the recovery of phenol-treated Escherichia coli. The effects observed varied with the concentration and the ionic species. In nutrient agar, Fe(3+) and Cr(3+) were generally beneficial but were toxic at 37 x 10(-4)m. Of the divalent ions tested, Ca(2+) and Mg(2+) usually gave higher counts in nutrient broth, except at a concentration of 9.25 x 10(-4)m, whereas the effect of Mn(2+) was rather variable. Two possible explanations are suggested to explain these effects. Toxic materials may be removed from the media by the precipitates formed on the addition of Fe(3+) or Cr(3+), or, in the case of the divalent ions, the integrity of the bacterial cell membranes may be maintained.     

Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.     

Kinetic linked-function analysis of the multiligand interactions on Mg(2+)-activated yeast pyruvate kinase.

The multiligand interactions governing the allosteric response of Mg(2+)-activated yeast pyruvate kinase (YPK) during steady-state turnover were quantitated by kinetic linked-function analysis. The substrate, PEP, the enzyme-bound divalent metal, Mg(2+), and the allosteric effector, FBP, positively influence each other's interaction with the enzyme in the presence of saturating concentrations of the second substrate, MgADP. The presence of Mg(2+) enhances the interaction of PEP and of FBP with YPK by -2.0 and -1.0 kcal/mol, respectively. The simultaneous interaction of PEP, Mg(2+), and FBP with YPK is favored by -4.1 kcal/mol over the sum of their independent binding free energies. The coupling free energies measured for Mg(2+)-activated YPK are weaker than the corresponding coupling free energies measured for Mn(2+)-activated YPK [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803], but are consistent with results of thermodynamic measurements with the Mg(2+)-YPK complex [Bollenbach, T. J., and Nowak, T. (2001) Biochemistry 36, 13088-13096]. A comparison of ligand binding data measured by kinetic and thermodynamic linked-function analyses reveals that the MgADP complex modulates both the binding of the other three ligands and the two- and three-ligand coupling interactions between the other three ligands. Enzyme-bound Mg(2+) does not influence the homotropic cooperativity in PEP binding to YPK. It is the MgADP complex that induces homotropic cooperativity in PEP binding. It is the enzyme-bound Mn(2+) that induces homotropic binding of PEP with Mn(2+)-activated YPK. These results lend support to the hypothesis that divalent metals modulate the interactions of ligands on YPK and that divalent metals play a role in regulation of the glycolytic pathway.     

Changing the metal ion selectivity of rabbit muscle enolase by mutagenesis: effects of the G37A and G41A mutations

During the reaction catalyzed by enolase, a mobile loop, residues 36-45, closes over the active site. In order to probe the role of this loop movement in catalysis, the glycines at positions 37 and 41 of rabbit muscle enolase (beta beta) have been mutated to alanines. The mutant forms-G37A and G41A-of enolase are both active, but have altered selectivity for divalent cations. G37A, when assayed with Mg(2+), has 12% the activity of the wild type. However, it is twice as active as wild type when assayed with Mn(2+), Zn(2+), or Co(2+). G41A has 4% the activity of the wild type with Mg(2+), is more active than wild type with Co(2+), and slightly less active than wild type with Mn(2+) and Zn(2+). The kinetic isotope effect for both mutants is greater than that of the wild type with all 4 divalent cations. These results indicate that the flexibility of this loop has subtle effects on catalytic activity.     

Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster

Integrin alpha(4)beta(7) mediates rolling adhesion in Ca(2+) and Ca(2+) + Mg(2+), and firm adhesion in Mg(2+) and Mn(2+), mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding sites present in integrin beta-subunit I-like domains. The middle, metal ion-dependent adhesion site (MIDAS) is required for both rolling and firm adhesion. One polar site, that adjacent to MIDAS (ADMIDAS), is required for rolling because its mutation results in firm adhesion. The other polar site, the ligand-induced metal binding site (LIMBS), is required for firm adhesion because its mutation results in rolling. The LIMBS mediates the positive regulatory effects of low Ca(2+) concentrations, whereas the ADMIDAS mediates the negative regulatory effects of higher Ca(2+) concentrations, which are competed by Mn(2+). The bipolar sites thus stabilize two alternative phases of adhesion.     

Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mn2+

The use of Mn(2+) as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg(2+) often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(pol)) in the presence of Mn(2+). The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg(2+) and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn(2+), the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn(2+), it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg(2+), highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn(2+) to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg(2+), this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn(2+) may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg(2+).     

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

We have analyzed the divalent cation specificity of poliovirus RNA-dependent RNA polymerase, 3D(pol). The following preference was observed: Mn(2+) > Co(2+) > Ni(2+) > Fe(2+) > Mg(2+) > Ca(2+) > Cu(2+), and Zn(2+) was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn(2+), 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg(2+). Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn(2+) was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn(2+), and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn(2+) as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-(32)P-end-labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg(2+). In the presence of Mn(2+), 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg(2+). Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation.     

Mechanistic insights into the retaining glucosyl-3-phosphoglycerate synthase from mycobacteria

Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.     

Site-specific effects of zinc on the activity of family II pyrophosphatase

Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn(2+)-containing metalloenzymes with two metal-binding subsites (M1 and M2) in the active site. These PPases can use a number of other divalent metal ions as the cofactor but are inactive with Zn(2+), which is known to be a good cofactor for family I PPases. We report here that the Mg(2+)-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn(2+), but the activity thereafter decays on a time scale of minutes. The activation of the Mn(2+)-form by Zn(2+) was slower but persisted for hours, whereas activation was not observed with the Ca(2+)- and apo-forms. The bound Zn(2+) could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn(2+) on PPase dimerization, the Zn(2+) binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn(2+) and EDTA were observed with the Mg(2+)- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn(2+) on the apo- and Mg(2+)-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not of HQ9 variant (modified M1 subsite) were similar to that for the Mn(2+)-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg(2+) and Mn(2+) at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn(2+) results from its binding to the M1 subsite; and (c) the subsequent inactivation of Mg(2+)-PPase results from Zn(2+) migration to the M2 subsite. The inability of Zn(2+) to activate apo-PPase suggests that Zn(2+) binds more tightly to M2 than to M1, allowing direct binding to M2. Zn(2+) is thus an efficient cofactor at subsite M1 but not at subsite M2.     

An isothermal titration calorimetry study of the binding of substrates and ligands to the tartrate dehydrogenase from Pseudomonas putida reveals half-of-the-sites reactivity

An isothermal titration calorimetric study of the binding of substrates and inhibitors to different complexes of tartrate dehydrogenase (TDH) from Pseudomonas putida was carried out. TDH catalyzes the nicotinamide adenine dinucleotide (NAD)-dependent oxidative decarboxylation of d-malate and has an absolute requirement for both a divalent and monovalent metal ion for activity. The ligands Mn(2+), meso-tartrate, oxalate, and reduced nicotinamide adenine dinucleotide (NADH) bound to all TDH complexes with a stoichiometry of 1 per enzyme dimer. The exception is NAD, which binds to E/K(+), E/K(+)/Mn(2+), and E/K(+)/Mg(2+) complexes with a stoichiometry of two per enzyme dimer. The binding studies suggest a half-of-the-sites mechanism for TDH. No significant heat changes were observed for d-malate in the presence of the E/K(+)/Mn(2+) complex, suggesting that it did not bind. In contrast, meso-tartrate does bind to E/K(+)/Mn(2+) but gives no significant heat change in the presence of E/Mn(2+), suggesting that K(+) is required for meso-tartrate binding. meso-Tartrate also binds with a large DeltaC(p) value and likely binds via a different binding mode than d-malate, which binds only in the presence of NAD. In contrast to all of the other ligands tested, the binding of Mn(2+) is entropically driven, likely the result of the entropically favored disruption of ordered water molecules coordinated to Mn(2+) in solution that are lost upon binding to the enzyme. Oxalate, a competitive inhibitor of malate, binds with the greatest affinity to E/K(+)/Mn(2+)/NADH, and its binding is associated with the uptake of a proton. Overall, with d-malate as the substrate, data are consistent with a random addition of K(+), Mn(2+), and NAD followed by the ordered addition of d-malate; there is significant synergism in the binding of NAD and K(+). Although the binding of meso-tartrate also requires enzyme-bound K(+) and Mn(2+), the binding of meso-tartrate and NAD is random.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

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.     

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)