Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase

Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co2+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the V for oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the pKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mg2+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity.     

Fluorescence characterization of the interaction of Al3+ and Pd2+ with Suwannee River fulvic acid in the absence and presence of the herbicide 2,4-dichlorophenoxyacetic acid

In an effort to understand the role of environmental metal ions in the interaction of charged pesticides with humic substances, a fluorescence study of the interaction of the widely-used herbicide 2,4-dichlorophenoxyacetic acid (DCPAA) with Al(3+) and Pd(2+) and Suwannee River fulvic acid (SRFA) was undertaken. Initial fluorescence experiments on binary solutions clearly indicated that both Al(3+) and Pd(2+) strongly interact with both SRFA and DCPAA when alone in solution with the metal ion. Titrations of SRFA with Al(3+) at pH values of 4.0, 3.0 and 2.0 revealed decreased degrees of fluorescence emission enhancement (at lambda(emission, max)=424 nm) with decreasing pH, consistent with the expected loss of rigidity in the SRFA-Al(3+) complexes formed as pH is lowered. In contrast, titrations of SRFA with Pd(2+) at all of these pH values resulted in significant fluorescence quenching. Al(3+) additions to solutions of DCPAA at pH values above the pK(a) (2.64) of DCPAA resulted primarily in significant changes in the wavelength of maximum emission (without significant quenching or enhancement of emission intensity), while Pd(2+) additions to DCPAA solutions resulted primarily in very significant fluorescence quenching. The DCPAA fluorescence results strongly support the formation of an Al(3+)-DCPAA complex at pH values above the pK(a) of DCPAA. The fluorescence results obtained for solutions of Pd(2+) and DCPAA are best explained by a collisional quenching mechanism, that is, energy transfer from excited DCPAA molecules to Pd(2+) following the collision of these two species in solution. Excitation-emission matrix plots obtained on ternary solutions (at environmentally-relevant pH 4.0) containing SRFA, DCPAA and metal ions (i.e., either Al(3+) or Pd(2+)) provides evidence (especially for systems containing Al(3+)) for the existence of ternary complexes between fulvic acid species, the herbicide DCPAA and metal ion, suggesting (at least at pH 4.0, where the predominant DCPAA species is negatively-charged) that metal ions may function to "bridge" negatively-charged fulvic acids to negatively-charged pesticides.     

Acid–base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M(2+) = Zn(2+), Cd(2+)), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H(+) in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH(2) group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximately 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S(-) unit (formation degree above 99.99% compared with that of N3). However, again a large degree of chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH(2) group (pK (a) = 12.65) is dramatically acidified (pK (a) approximately 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleoside.     

Effects of pH and metal ions on antioxidative activities of catechins

The Effects of pH on antioxidative activities of catechol, pyrogallol, and four catechins, and effects of metal ions (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, and Zn2+) on antioxidative activities of (-)-epigallocatechin gallate (EGCG) were studied by an oxygen electrode method. The antioxidative activities of catechins were high and constant at pH 6-12, but decreased in acidic and strong alkaline solutions. Copper(II) ion the most strongly increased the antioxidative activity of EGCG among these metal ions examined, but iron(II) ion largely inhibited the antioxidative activity of EGCG. These effects are discussed considering the formation of metal complexes with catechins and the change in oxidation potentials.     

The Fe(III)Zn(II) form of recombinant human purple acid phosphatase is not activated by proteolysis

The kinetics and spectroscopic properties of the single polypeptide and proteolytically cleaved form of recombinant Fe(3+)Fe(2+) human purple acid phosphatase (recHPAP) exhibit significant differences, primarily due to a difference in pK(es,1) (the value of an acid dissociation constant of the ES complex). These differences are due to the presence or absence, respectively, of an interaction between an aspartate residue in an exposed loop of the protein and one or more active site residues. To further explore the origin of these differences, the ferrous ion of recHPAP has been replaced by zinc. Analysis of the reconstituted Fe(3+)Zn(2+)recHPAP reveals an unexpected catalytic activity versus pH profile, in that the optimal pH is 6.3, similar to that of the proteolytically cleaved form (6.5). Moreover, replacement of the ferrous ion by zinc increases the turnover number more than 10-fold; the pK(es) values are also shifted as expected for the change in the divalent metal ion. Although the EPR spectra of both single polypeptide and proteolytically cleaved Fe(3+)Zn(2+)-recHPAP are independent of pH over the range 4.5-6.2, the visible spectrum of Fe(3+)Zn(2+)-recHPAP is pH dependent. These results suggest that the properties and environment of the divalent metal are important in determining the catalytic properties of mammalian PAPs, and in particular that a solvent molecule coordinated to the divalent metal ion may play a critical role in the catalytic cycle of these enzymes.     

Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological system

To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hückel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C.     

The pH variation of steady-state kinetic parameters of site-specific Co(2+)-reconstituted liver alcohol dehydrogenase. A mechanistic probe for the assignment of metal-linked ionizations

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.     

Spectral control of metal admixtures in tetracycline]

Analysis of circular dichroism spectra made it possible to offer a method for estimation of tetracycline solutions contamination with metal ions. By its sensitivity the method is much superior to the spectrophotometric one used at present for determination of the antibiotic purity. In the latter method formation of complexes with metals is traced by batochromic displacement of the absorption spectra. The new method is rapid, relatively selective and requires comparatively small quantities of the substance for the analysis, which provides its use under both laboratory and manufacture conditions. The method is based on identification of the circular dichroism spectra of tetracycline complexes with metals in the long wavelength region. The presence of the circular dichroism concervative bands with strictly defined extremums in the spectra of tetracycline low acid solutions contaminated by multiply charged metal ions allowed vs. the circular dichroism spectra of pure tetracycline sample to conclude that the solution contained admixtures and to suggest their nature. It was shown that the charge, ion radius and tetracycline:metal relation were the factors defining the mark and location of the dichroism band extremums. At lambda(extr)-410-415 nm the tetracycline complexes with light metal ions such as Mg2+, Al3+ and Ca2+ were detected by the circular dichroism negative band in the spectra, while the complexes with heavy metal ions such as Sc3+, Sr3+, Cu3+, Cd3+, Ba2+, Y3+ and the cerium subgroup lanthanides were detected by the circular dichroism positive band. The tetracycline complexes with the lanthanides of the second half of the yttrium subgroup (Ho(3+)-Lu3+) were characterized by the presence of the circular dichroism minimum at lambda(min)-425 nm. When the tetracycline concentration was above 1.5 x 10(-3) M, multiligand complexes with circular dichroism negative extremum at lambda(min)-400 nm formed.     

Metal ion-carbonyl oxygen recognition in complexes of acetyl phosphate

Studies on acetyl phosphate (AcP2-), one of the so-called 'energy-rich' mixed-acid anhydrides, are summarized. Based on stability constants determined by potentiometric pH titrations in aqueous solution, it is shown that the M(AcP) complexes of Ca2+, Mg2+, Mn2+, Cu2+, and Zn2+ are more stable than is expected from the basicity of the phosphate group of AcP2-. This observed stability increase is attributed to an additional interaction of the already phosphate-coordinated metal ion (M2+) with the carbonyl oxygen of the anhydride unit. These conclusions are corroborated by the properties of the complexes of the hydrolysis-stable acetonylphosphonate (AnP2-). The formation degrees of the various six-membered chelates occurring in the M(AcP) and M(AnP) systems are presented and evidence is given that these chelates persist in mixed ligand complexes and that their formation degree is promoted by a low solvent polarity. The biological relevance of these results regarding carbonyl oxygen-metal ion interactions is briefly indicated.     

Site-directed mutagenesis study of the microenvironment characteristics of Lys213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn(2+) as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. Nature Struct. Biol. 4 (1997) 990-994] shows that Lys(213) is one of the ligands to Mn(2+) at the enzyme active site. Coordination of Mn(2+) to a lysyl residue is infrequent and suggests a low pK(a) value for the epsilon-NH(2) group of Lys(213). In this work, we evaluate the role of neighboring Phe(416) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe416Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn(2+), suggesting an increase of the pK(a) of Lys(213). A study of the effect of pH on K(m) for Mn(2+) indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK(a) of 7.1+/-0.2, compatible with the low pK(a) expected for Lys(213). This pK(a) value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys(213) microenvironment. Theoretical calculations of the pK(a) of Lys(213) indicate a value of 6.5+/-0.9, and it increases to 8.2+/-1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol(-1) in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe(416) to Arg(70), a residue previously shown to be important for PEP binding.     

1H Fourier transform NMR studies of insulin: coordination of Ca2+ to the Glu(B13) site drives hexamer assembly and induces a conformation change

1H Fourier transform NMR investigations of metal ion binding to insulin in 2H2O were undertaken as a function of pH* to determine the effects of metal ion coordination to the Glu(B13) site on the assembly and structure of the insulin hexamer. The C-2 histidyl regions of the 1H NMR spectra of insulin species containing respectively one Ca2+ and two Zn2+/hexamer and three Cd2+/hexamer have been assigned. Both the Cd2+ derivative (In)6(Cd2+)2Cd2+, where two of the Cd2+ ions are coordinated to the His(B10) sites and the remaining Cd2+ ion is coordinated to the Glu(B13) site [Sudmeier, J.L., Bell, S.J., Storm, M. C., & Dunn, M.F. (1981) Science (Washington, D.C.) 212, 560], and the Zn2+-Ca2+ derivative (In)6-(Zn2+)2Ca2+, where the two Zn2+ ions are coordinated to the His(B10) sites and Ca2+ ion is coordinated to the Glu(B13) site, give spectra in which the C-2 proton resonances of His(B10) are shifted upfield relative to metal-free insulin. Spectra of insulin solutions (3-20 mg/mL) containing a ratio of In:Zn2+ = 6:2 in the pH* region from 8.6 to 10 were found to contain signals both from metal-free insulin species and from the 2Zn-insulin hexamer, (In)6(Zn2+)2. The addition of either Ca2+ (in the ratio In:Zn2+:Ca2+ = 6:2:1) or 40 mM NaSCN was found to provide sufficient additional thermodynamic drive to bring about the nearly complete assembly of insulin hexamers. Cd2+ in the ratio In:Cd2+ = 6:3 also drives hexamer assembly to completion. We postulate that the additional thermodynamic drive provide by Ca2+ and CD2+ is due to coordination of these metal ions to the Glu(B13) carboxylates of the hexamer. At high pH*, this coordination neutralizes the repulsive Coulombic interactions between the six Glu(B13) carboxylates and forms metal ion "cross-links" across the dimer-dimer interfaces. Comparison of the aromatic regions of the 1H NMR spectra for (In)6(Zn2+)2 with (In)6(Zn2+)2Ca2+, (In)6(Cd2+)2Cd2+, and (In)6(Cd2+)2Ca2+ indicates that binding of either Ca2+ or Cd2+ to the Glu(B13) site induces a conformation change that perturbs the environments of the side chains of several of the aromatic residues in the insulin structure. Since these residues lie on the monomer-monomer and dimer-dimer subunit interfaces, we conclude that the conformation change includes small changes in the subunit interfaces that alter the microenvironments of the aromatic rings.     

The role of metal on imide hydrolysis: metal content and pH profiles of metal ion-replaced mammalian imidase

Imidase catalyzes the hydrolysis of a variety of imides. The removal of metal from imidase eliminates its activity but does not affect its tetrameric and secondary structure. The reactivation of the apoenzyme with transition metal ions Co(2+), Zn(2+), Mn(2+), and Cd(2+) shows that imidase activity is linearly dependent on the amount of metal ions added. Ni(2+) and Cu(2+) are also inserted, one per enzyme subunit, into the apoimidase, but do not restore imidase activity. Enzyme activity with different metal replaced imidase varies significantly. However, the changes of the metal contents do not appear to affect the pK(a)s obtained from the bell-shaped pH profiles of metal reconstituted imidase. The metal-hydroxide mechanism for imidase action is not supported based on the novel findings from this study. It is proposed that metal ion in mammalian imidase functions as a Lewis acid, which stabilizes the developing negative charge of imide substrate in transition state.     

Characterization and implications of Ca2+ binding to pectate lyase C

Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.     

Crystal structures and spectroscopic characterization of galactitol complexes of trivalent lanthanide and divalent alkaline earth chlorides

Crystal structures and FT-IR spectra of metal ion-galactitol (C6H14O6, the ligand here abbreviated as L) complexes: 2LaCl3*C6H14O6*10H2O and SrCl2*C6H14O6 complexes are reported. Crystal data of lanthanide chlorides (La3+, Nd3+, Sm3+, Eu3+, Tb3+)-galactitol complexes and alkaline earth chlorides (Ca2+, Sr2+)-galactitol complexes published earlier are summarized. Unlike other lanthanide ion-galactitol complexes (2MCl3*C6H14O6*14H2O), lanthanum ions give rise to two different structures: LaCl3*C6H14O6*6H2O (LaL1) and 2LaCl3*C6H14O6*10H2O (LaL2). Sr2+-galactitol complexes also crystallized with two structures: SrCl2*C6H14O6*4H2O (SrL1) and SrCl2*C6H14O6 (SrL2). These metal ions thus give different coordination structures with galactitol. The crystal structures and FT-IR spectra of lanthanide ion and alkaline earth ion-galactitol complexes were integrated to interpret the coordination modes of different metal ions. Similar IR spectra demonstrate the same coordination modes of the complexes.     

Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme-Mn(2+)-isocitrate complex, Asp(252), Asp(275), and Asp(279) were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The K(m) values for isocitrate and for Mn(2+) are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the K(m)'s are not changed appreciably. Mutants at position 279 exhibit only a small change in K(m) for isocitrate. These results indicate that Asp(252) and Asp(275) are ligands of enzyme-bound Mn(2+)and influence the binding of Mn(2+)-isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn(2+). The pK(aes)'s of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the pK(aes) of D279C is a little lower (about 4.7). These findings suggest that the V(max)'s of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp(252), Asp(275), nor Asp(279) acts as the general base in the enzymatic reaction. For wild-type enzyme, the pK(aes) varies with the metal ion used with Mg(2+) > Cd(2+) > Mn(2+) > Co(2+), similar to the order of the pK's for these four metal-bound waters. We therefore attribute the pH dependence of V(max) to the deprotonation of the metal-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase.     

Extent of metal ion-sulfur binding in complexes of thiouracil nucleosides and nucleotides in aqueous solution

Previously published stability constants of several metal ion (M2+) complexes formed with thiouridines and their 5'-monophosphates, together with recently obtained log K(M(U))(M) versus pK(U)(H) plots for M2+ complexes of uridinate derivatives (U-) allowed now a quantitative evaluation of the effect that the exchange of a (C)O by a (C)S group has on the stability of the corresponding complexes. For example, the stability of the Ni2+, Cu2+ and Cd2+ complexes of 2-thiouridinate is increased by about 1.6, 2.3, and 1.3 log units, respectively, by the indicated exchange of groups. Similar results were obtained for other thiouridinates, including 4-thiouridinate. The structure of these complexes and the types of chelates formed (involving (N3)- and (C)S) are discussed. A recently advanced method for the quantification of the chelate effect allows now also an evaluation of several complexes of thiouridinate 5'-monophosphates. In most instances the thiouracilate coordination dominates the systems, allowing only the formation of small amounts of phosphate-bound isomers. Among the complexes studied only the one formed by Cu2+ with 2-thiouridinate 5'-monophosphate leads to significant amounts of the macrochelated isomer, which means that in this case Cu2+ is able to force the nucleotide from the anti to the syn conformation, allowing thus metal ion binding to both potential sites and this results in the formation of about 58% of the macrochelated isomer. The remaining 42% are species in which Cu2+ is overwhelmingly coordinated to the thiouracilate residue; Cu2+ binding to the phosphate group occurs in this case only in trace amounts.     

O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145

The active site cysteine of human O(6)-alkylguanine-DNA alkyltransferase (hAGT), Cys145, was shown to be highly reactive with model electrophiles unrelated to substrates, including 1-chloro-2,4-dinitrobenzene. The high reactivity suggested that the Cys145 thiolate anion might be stable at neutral pH. The pK(a) was estimated from plots of UV spectra (A(239)) and reactivity toward 4,4'-dithiopyridine vs pH. The estimated pK(a) for hAGT was 4-5, depending upon the method used, and near that of the extensively characterized papain Cys25. Rates of reaction with 4,4'-dithiopyridine were similar for the thiolate forms of hAGT, papain, glutathione, and the bacterial hAGT homologue Ogt (the pK(a) of the latter was 5.4). Bound Zn(2+) has previously been shown to be required for the catalytic activity of hAGT (Rasimas, J. J. et al. (2003) Biochemistry 42, 980-990). Zn(2+) was shown to be required for the low pK(a) of hAGT. The high reactivity of hAGT Cys145 is postulated to be important in normal catalytic function, in cross-linking reactions involving bis-electrophiles, and in inhibition of the DNA repair function of hAGT by electrophiles.     

Different metal-binding properties of the two sites of human transferrin.

Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.     

Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues

Transition metal ions bind to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides and slow the light-induced electron and proton transfer to the secondary quinone, Q(B). We studied the properties of the metal ion-RC complex by measuring the pH dependence of the dissociation constant and the stoichiometry of proton release upon ligand formation. We investigated the mechanism of inhibition by measuring the stoichiometry and kinetics of flash-induced proton binding, the transfer of (first and second) electrons to Q(B), and the rate of steady-state turnover of the RC in the absence and presence of Cd(2+) and Ni(2+) on a wide pH range. The following results were obtained. (1) The complexation of transition metal ions Cd(2+) and Ni(2+) with the bacterial RC showed strong pH dependence. This observation was explained by different (pH-dependent) states of the metal-ligand cluster: the complex formation was strong when the ligand (Asp and His residues) was deprotonated and was much weaker if the ligand was partly (or fully) protonated. A direct consequence of the model was the pH-dependent proton release upon complexation. (2) The retardation of transfer of electrons and protons to Q(B) was also strongly pH-dependent. The effect was large in the neutral pH range and decreased toward the acidic and alkaline pH values. (3) Steady-state turnover measurements indicated that the rate of the second proton transfer was much less inhibited than that of the first one, which became the rate-limiting step in continuous turnover of the RC. (4) Sodium azide partly recovered the proton transfer rate. The effect is not due to removal of the bound metal ion by azide but probably by formation of a proton-transporting azide network similarly as water molecules may build up proton pathways. (5) We argue that the inhibition comes mainly from pK(a) shifts of key protonatable residues that control the proton transfer along the H-bond network to Q(B). The electrostatic interaction between the metal ion and these residues may result in acidic pK(a) shifts between 1.5 and 2.0 that account for the observed retardation of the electron and proton transfer.     

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.