25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase.

25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral properties of Co(II)- and Ni(II)-activated rabbit muscle pyruvate kinase.

Stoichiometry, kinetics, and optical properties of rabbit muscle pyruvate kinase activated with Co(II), Ni(II), Mg(II), and Mn(II) were studied. The stoichiometry of metal binding to enzyme was found to be 4 metal ions per tetrameric enzyme for Co(II) and Ni(II) by carrying out circular dichroic titrations. Cu(II) and Fe(II) were inactive. Ca(II) and Zn(II) were not activating, and were inhibitory with respect to all of the active cations. The temperature dependence of the optimal velocity is similar for all activating metals. The pH rate profiles suggest that there are two classes of enzyme activation by metal ions. Mg(II) and Mn(II) are quite similar to each other while Co(II) and Ni(II) are different from them but similar to each other. Absorption, natural, and magnetic CD in the visible region were used to probe the environment of the activating divalent cation in Ni(II)- and Co(II)-activated pyruvate kinase and their complexes with substrates and inhibitors...     

Synthesis and characterization of metal complexes of Calcein Blue: Formation of monomeric, ion pair and coordination polymeric structures

A series of metal complexes containing the 4-methylumbelliferone-8-methyleneiminodiacetic acid (H₃muia, also named as Calcein Blue) has been synthesized and characterized. Complexes of Cu(II), Ni(II), Mn(II), Zn(II) and Mg(II) have been structurally characterized while Ca(II) and Al(III) complexes by elemental analysis and thermogravimetry. The Cu(II) and Ni(II) complexes are neutral and mononuclear in the solid state. Interestingly, the Mn(II), Zn(II) and Mg(II) muia complexes exist as ion-pairs containing hydrated or solvated metal cation and dimeric metal(II) muia anions. Owing to the presence of hydrogen-bond donors and acceptors in the ligand, hydrogen bonding interactions are dominant along with π-π stacking in their solid-state structures. The solid-state fluorescence studies indicate that the family of muia complexes exhibit comparable emission properties as in solution state, in which only main group and post-transition complexes show bright blue fluorescence while transition metal complexes do not fluoresce.     

Structure of the bis divalent cation complex with phosphonoacetohydroxamate at the active site of enolase.

Phosphonoacetohydroxamate (PhAH) is a tight-binding (Ki = 15 pM) inhibitor of enolase that is believed to mimic the aci-carboxylate form of the intermediate carbanion in the reaction [Anderson, V. E., Weiss, P. M., & Cleland, W. W. (1984) Biochemistry 23, 2779]. Electron paramagnetic resonance (EPR) spectroscopy of Mn2+ has been used to map sites of interaction of PhAH with the two divalent cations at the active site of enolase from bakers' yeast. EPR spectra of enolase-PhAH complexes containing two Mn2+ bound at the active site contain multiple fine structure transitions each with a 45-G 55Mn hyperfine spacing that is a characteristic of spin exchange coupled pairs of Mn2+. Magnetically dilute complexes were obtained by preparation of specific Mg2+/Mn2+ hybrid complexes by manipulating the order of addition of the divalent metal species. Thus, Mn2+ was placed in the higher affinity site by addition of 1 equiv of Mn2+ to a solution of enolase and PhAH, followed by addition of 1 equiv of Mg2+. Reversing the order of addition of Mg2+ and Mn2+ placed Mn2+ in the lower affinity site. Regiospecifically 17O-labeled forms of PhAH were prepared, and the binding of the functional groups on PhAH to Mn2+ at the two metal ion sites was determined from the presence or absence of 17O superhyperfine coupling in the EPR signals. The hydroxamate oxygen is a ligand of Mn2+ at the higher affinity site, a phosphonate oxygen is a ligand of Mn2+ at the lower affinity site, and the carbonyl oxygen is a mu-O bridge of the two metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of Na(+) to apo-enolase permits the binding of substrate

Enolase from rabbit muscle (betabeta-enolase) is inactivated by NaClO(4). Enolase free of divalent cations is more susceptible to inactivation by NaClO(4) than is enolase in the presence of Mg(2+). We find that substrate protects apo-enolase against inactivation, indicating that substrate can bind to enolase in the absence of a divalent cation. This binding is not due to contamination by trace levels of divalent cations since (1) it occurs even in the presence of EDTA or EGTA and (2) metal analysis by ICP (inductively coupled plasma) mass spectrometry did not reveal sufficient contamination to account for the protection. The binding of PGA to apo-enolase did require Na(+). When TMAClO(4) was used instead of NaClO(4), there was no protection by PGA. Protection was restored when TMAClO(4) plus NaCl were used. The inactivation of apo-enolase by NaClO(4) is due to dissociation into inactive monomers. We conclude that Na(+) binds to apo-enolase, permitting substrate to then bind. Of the three known Me(2+) binding sites on enolase, we believe the most likely binding site for Na(+) is the carboxylate cluster of site 1, the highest affinity site of enolase.     

Activation of rabbit kidney fructose diphosphatase by Mg-EDTA, Mn-EDTA and Co-EDTA complexes

Purified rabbit kidney fructose diphosphatase requires both a free cation and a metal-chelate when assayed at pH 8 or below. In the presence Mg²⁺ or Mn²⁺, effective metal chelates were Mn(II)-EDTA, Mg(II)-EDTA, and Co(III)-EDTA. With Mg²⁺ as the cation the affinity of the enzyme for Mn(II)-EDTA or Mg(II)-EDTA was approximately the same, and 300-fold greater than that for Co(III)-EDTA.Activation of the enzyme by the very stable Co(III)-EDTA complex, as well as failure of an ionophore antibiotic to replace EDTA as activator, exclude the possibility that the effects of EDTA are due to removal of metal inhibitors.Inhibition of fructose diphosphatase by Ca²⁺ was competitive with Mg²⁺, and noncompetitive with Mg(II)-EDTA, or Co(III)-EDTA. Conversely inhibition by Zn(II)-EDTA was competitive with Mg(II)-EDTA and noncompetitive with free Mg²⁺. The data suggest that the free metals bind to one site on the enzyme while the metal-EDTA chelates bind to a second site.     

Magnetic resonance and kinetic studies of the partial complex and Component I subunit forms of Salmonella typhimurium anthranilate synthase

Metal ion interactions of the monofunctional partial complex of Salmonella typhimurium anthranilate synthase were investigated using kinetic, NMR, and EPR methods. Mn2+ activates AS-partial complex in place of Mg2+, with a Km of 0.08 microM for Mn2+ and of 3.5 microM for Mg2+ in glutamine-dependent anthranilate synthase activity. The kinetics indicated that the metal interacts at the active site with chorismate, not glutamine. EPR and NMR water proton relaxation rate (PRR) studies supported this conclusion. EPR binding analysis showed that chorismate dramatically tightens Mn2+ binding by the partial complex. PRR experiments indicated that stoichiometric amounts of chorismate cause a substantial decrease in the enhancement of water relaxation by Mn2+, while millimolar amounts of glutamine have no effect. Analysis of the frequency dependence of water proton relaxation rates yielded dipolar correlation times of 2.5 x 10(-9) s and 4.1 x 10(-9) s for the Mn2+-partial complex and Mn2+-partial complex-chorismate complexes, respectively. These studies also indicated that chorismate binding reduces the number of fast-exchanging water molecules on enzyme-bound Mn2+ from 1 to 0.25. PRR experiments with the native bifunctional anthranilate synthase-phosphoribosyltransferase enzyme indicated the existence of additional Mn2+-binding sites which presumably function to activate the phosphoribosyltransferase activity of the Component II subunit.     

Kinetic analysis of the effect of zinc ion on the inorganic pyrophosphatase reaction.

A kinetic study is presented in which the effect of Zn(II) on yeast inorganic pyrophosphatase was quantitatively determined. A dual role model for metal ion effect, previously determined for the Mg(II)-pyrophosphatase system (O. A. Moe and L. G. Butler, 1972, J. Biol. Chem.247, 7308–7315), was applied successfully to the analysis of the kinetics for Zn(II)-pyrophosphatase and Zn(II), Mg(II)-pyrophosphatase systems. The model, assigning an activator role to free Zn(II) ion and a substrate role to the Zn(II)-pyrophosphate complex, gave an excellent fit to the data. Inhibition of the Mg(II)-pyrophosphatase system by Zn(II) was analyzed by a model in which competitive binding of the Mg(II)-pyrophosphate and Zn(II)-pyrophosphate complexes occurred at the enzyme active site, with both complexes undergoing reaction at different rates. Relative maximal velocities and enzymeligand dissociation constants for the Zn(II)-pyrophosphate complex were determined for the cases where the metal ion activator role was fulfilled by Zn(II) and Mg(II), respectively. The maximal velocity parameter showed a dependence on the nature of the activator metal ion, demonstrating that the role of the latter is associated both with the process of substrate binding and with the mechanism of catalysis. Values for all kinetic parameters are reported for an ionic strength of 0.2, pH 7.0, and 25.0 °C.     

The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.     

Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation [Baek, Y. H., & Nowak, T. (1982) Arch. Biochem. Biophys. 217, 491-497]. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EPR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Addition of Mg(II) to solutions of enzyme, Mn(II), ATP, and oxalate sharpens the EPR signals for the enzyme-bound Mn(II). Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of 17O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the gamma-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling form 17O regiospecifically incorporated into the gamma-phosphate group of ATP. By contrast, 17O in the alpha-phosphate or in the beta-phosphate groups of ATP does not influence the spectrum. Experiments in 17O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Effect of metal ions on the fluorescence of leucine aminopeptidase and its dansyl-peptide substrates

The effect of Cu(II), Ni(II), Zn(II), Mg(II), and Mn(II) on the fluorescence of porcine kidney cytosol leucine aminopeptidase and three of its dansyl(Dns) peptide substrates, Leu-Gly-NHNH-Dns, Leu-Gly-NH(CH2)2NH-Dns, and Leu-Gly-NH(CH2)6NH-Dns, has been investigated. These five metal ions were chosen for study because each binds to the regulatory metal binding site of leucine aminopeptidase. Since the binding is relatively weak, kinetic studies of the different metalloderivatives of the enzyme are normally carried out in the presence of large molar excesses of these metal ions that can potentially affect both the enzyme and substrate. The fluorescence of all of the dansyl-peptides, as well as several other dansyl species, is quenched by Ni(II) and Cu(II), but not by Mg(II), Mn(II), or Zn(II). The absorption spectra of these dansyl substrates are also perturbed by Ni(II) and Cu(II). The rate at which maximal quenching for some dansyl species is attained after mixing with Ni(II) and Cu(II) is slow and the quenching is reversed on addition of EDTA. These results indicate that the quenching is the result of complex formation between the fluorophores and these metal ions. The association constants for the metal complexes have been determined from Stern-Volmer plots. In addition to complex formation, Ni(II) and Cu(II) cause the degradation of Leu-Gly-NHNH-Dns through a two step mechanism involving loss of dansic acid. Ni(II) and Cu(II) also partially quench the fluorescence of leucine aminopeptidase through contact with its surface accessible Trp residues. These observations indicate that care must be taken in stopped flow fluorescence studies of reactions between this enzyme and its dansyl substrates to avoid adverse effects brought about by Ni(II) and Cu(II).     

Free metal ion depletion by "Good's" buffers. III. N-(2-acetamido)iminodiacetic acid, 2:1 complexes with zinc(II), cobalt(II), nickel(II), and copper(II); amide deprotonation by Zn(II), Co(II), and Cu(II)

Potentiometric, visible, infrared, electron spin, and nuclear magnetic resonance studies of the complexation of N-(2-acetamido)iminodiacetic acid (H2ADA) by Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) are reported. Ca(II) and Mg(II) were found not to form 2:1 ADA2- to M(II) complexes, while Mn(II), Cu(II), Ni(II), Zn(II), and Co(II) did form 2:1 metal chelates at or below physiological pH values. Co(II) and Zn(II), but not Cu(II), were found to induce stepwise deprotonation of the amide groups to form [M(H-1ADA)4-(2)]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed on the basis of various spectral data.     

Stopped-flow cryoenzymological investigation of the pre-steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns by leucine aminopeptidase

Stopped-flow fluorescence experiments have been carried out to study the steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] by porcine kidney cytosol leucine aminopeptidase (LAP) in 50% v/v methanol/buffer solution at ambient temperature and the pre-steady-state kinetics of this reaction in the -35 to 0 degrees C temperature range. Experiments have been carried out on LAP species containing Mg(II), Mn(II), Cu(II), Ni(II), Zn(II), and no metal ion at the regulatory metal binding site. At ambient temperatures, the stopped-flow fluorescence changes observed on hydrolysis of the substrate have been used to measure the steady-state kinetic parameters kcat and KM. The results show that 50% v/v methanol lowers the values of kcat from 2- to 12-fold compared to the reactions in the absence of methanol for all of the metallo-LAP, but that the values of KM are essentially unaffected. The pre-steady-state reactions carried out under nonturnover conditions at -35 degrees C reveal a new relaxation for LAP species with Ni(II), Cu(II), and Zn(II) in the regulatory site. The value of kobsd for this relaxation reaches a plateau at high substrate concentrations, and the magnitude of its fluorescence change at a fixed concentration of substrate is proportional to the enzyme concentration. Thus, this relaxation corresponds to the production and decay of a new enzyme-substrate intermediate not observed at higher temperatures whose fluorescence differs from that of the succeeding intermediate that is normally seen above -26 degrees C.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites

The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.     

113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.