Chemical modification of carbonic anhydrase II with acrolein

We have reacted acrolein with human carbonic anhydrase II using conditions reported to result in maximal formylethylation of exposed histidine and lysine residues (Pocker, Y., and Janji?, N. (1988) J. Biol. Chem. 263, 6169-6176). Pocker and Janji? proposed that the decrease by 95-98% in the steady-state turnover number for the hydration of CO2 caused by this chemical modification is due predominantly to the alkylation of one residue, the imidazole side chain of histidine 64. We measured the rate of 18O exchange between CO2 and water catalyzed by these enzymes at chemical equilibrium using membrane inlet mass spectrometry. The catalyzed rate of interconversion of CO2 and HCO3- at chemical equilibrium was the same for the acrolein-modified and the unmodified carbonic anhydrases, but the rate of release of 18O-labeled water from the active site had decreased by as much as 85% for the acrolein-modified enzyme. The 18O-exchange kinetics catalyzed by the acrolein-modified carbonic anhydrase II was similar to that catalyzed by a mutant human carbonic anhydrase II in which histidine at residue 64 was replaced with alanine. Moreover, modification of this mutant carbonic anhydrase II with acrolein did not alter to a significant extent its 18O-exchange pattern. These results support the proposal of Pocker and Janji? and the suggested role of histidine 64 in carbonic anhydrase II as a proton shuttle residue that transfers a proton from zinc-bound water to buffer in solution.     

Origin of viscosity effects in carbonic anhydrase catalysis. Kinetic studies with bulky buffers at limiting concentrations

In our earlier paper we showed that the rates of CO2 hydration and HCO3- dehydration catalyzed by the high-activity form of mammalian erythrocyte carbonic anhydrase (CA II) were dependent on solution viscosity increase and that the effect was linked to some kind of proton-transfer-related event [Pocker, Y., & Janji?, N. (1987) Biochemistry 26, 2597-2606]. In order to further elucidate the source of the observed viscosity effect, the dependence of kcat and Km for CA II catalyzed HCO3- dehydration at pH 5.90 on sucrose-induced viscosity increase was investigated at several concentrations of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, including the very low buffer concentration region (less than 10 mM) where the proton transfer between the shuttle group on the enzyme and buffer becomes rate limiting. In all examined cases, kcat steadily decreased with added sucrose while Km remained independent of the viscosity increase. The extent to which this reaction was dependent on viscosity was found to be constant, within experimental error, over the entire range of MES buffer concentrations studied (1-20 mM). Furthermore, the viscosity effect was qualitatively and quantitatively the same when an exceptionally large buffer (i.e., bovine serum albumin) was used instead of the more commonly used biological buffer (i.e., MES).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic 13C NMR investigations of substrate interaction and catalysis by cobalt(II) human carbonic anhydrase I

Using 13C NMR spectroscopy, we have further investigated the binding of HCO3- in the active site of an artificial form of human carbonic anhydrase I in which the native zinc is replaced by Co(II). The Co(II) enzyme, unlike all other metal-substituted derivatives, has functional properties closely similar to those of the native zinc enzyme. By measuring the spin-lattice relaxation rate and the line width for both the CO2 and HCO3- at two field strengths, we have determined both the paramagnetic effects that reflect substrate binding and the exchange effects due to catalysis at chemical equilibrium. The following are the results at 14 degrees C and pH 6.3 (1) HCO3- is bound in the active site of the catalytically competent enzyme with the 13C of the HCO3- located 3.22 +/- 0.02 A from the Co(II); (2) the apparent equilibrium dissociation constant for the bound HCO3- is 7.6 +/- 1.5 mM, determined by using the paramagnetic effects on the line widths, and 10 +/- 2 mM, determined by using the exchange effects; (3) the lifetime of HCO3- bound to the metal is (4.4 +/- 0.4) X 10(-5) s; (4) the overall catalyzed CO2 in equilibrium HCO3- exchange rate constant of the Co(II) enzyme is (9.6 +/- 0.4) X 10(3) s-1; (5) the electron spin relaxation time of the Co(II), determined by using paramagnetic effects on the bound HCO3-, is (1.1 +/- 0.1) X 10(-11) s. The data did not provide any direct information on the binding of CO2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.     

Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA.

Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of the steady-state kinetic and proton relaxation rate properties of the Mn(II)-containing enzyme

The essential Zn(II) in bovine liver dihydropyrimidine amidohydrolase (DHPase) was removed by incubation with 2,6-dipicolinic acid and replaced with Mn(II). Electron paramagnetic resonance studies of Mn(II) binding show that there are four binding sites per tetramer, and the dissociation constant at pH 7.5 is 13.5 microM. The substitution of Mn(II) for Zn(II) increases the specific activity of the enzyme approximately sixfold but has only a small effect (twofold increase) on the Km for 5-bromo-5,6-dihydrouracil (BrH2Ura). The pH dependence of the catalytic properties of Mn(II)-DHPase is the same as for the Zn(II) enzyme (Lee, M., Cowling, R., Sander, E., and Pettigrew, D. (1986) Arch. Biochem. Biophys. 248, 368-378). The pH dependence is well described in terms of the ionization of a single group with a pK of about 6 in the free enzyme. The ionization of this group is required for catalytic activity. The substitution of Mn(II) for Zn(II) does not affect the pH dependence of DHPase catalysis and therefore strongly suggests that the ionizable group is an amino acid residue at or near the active site, rather than a metal-bound water molecule. The pH dependence of the enhancement of the paramagnetic effect of the DHPase-Mn complex on the relaxation rate of the solvent water protons also is well described in terms of the ionization of a single group with a pK of about 6. Ionization of the group which is involved in catalysis also perturbs the environment of the bound Mn(II). The ionization of the active site group does not affect the number of exchangeable water molecules but does affect the symmetry of the environment of the bound Mn(II) and its electron relaxation.     

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.     

Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitrated in vitro and potentially in vivo by peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant and Escherichia coli Mn-SOD with a second order rate constant of 1.0 +/- 0.2 x 10(5) and 1.4 +/- 0.2 x 10(5) m(-)1 s(-)1 at pH 7.47 and 37 degrees C, respectively. The E. coli apoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <10(4) m(-)1 s(-)1 for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e. manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 +/- 0.1 x 10(4) m(-)1 s(-)1 and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.     

Carbamate formation on tubulin: CO2/bicarbonate buffers protect tubulin from inactivation by reductive methylation and carbamoylation and promote microtubule assembly at alkaline pH

Carbamoylation and reductive methylation of tubulin have been shown previously to inhibit microtubule assembly, probably by attack on essential internal lysine residues [Mellado, W., Slebe, J., & Maccioni, R.B. (1982) Biochem. J. 203, 675-681; Szasz, J., Burns, R., & Sternlicht, H. (1982) J. Biol. Chem. 257, 3697-3704]. We show first that this inhibition is blocked by the presence of HCO3-/CO2 buffer at physiological concentrations during the carbamoylation or reductive methylation. Under conditions that block assembly, the amount of radiolabeled cyanate or formaldehyde incorporated by these reactions in the absence of HCO3-/CO2 was approximately four carbamoyl or five methyl groups in a ratio of approximately 1.7 alpha chain/beta chain. In the presence of HCO3-/CO2, the formaldehyde incorporation is decreased roughly 0.5 mol in each of the alpha and beta chains, and cyanate incorporation, roughly 1.0 mol/mol of alpha or beta monomer. These results are consistent with the hypothesis that CO2 competed with formaldehyde or cyanate for uncharged amino groups and led to the reversible formation of carbamates. The complete antagonism of the inhibition of microtubule assembly by reductive methylation by CO2, even though the number of methyl groups incorporated was reduced by only 0.5 mol/tubulin monomer, was consistent with the possibility that reductive methylation opened up additional residues for attack. Indeed, using an adaptation of the method of Gros et al. for measurement of carbamates [Gros, G., Forster, R.E., & Lin, L. (1976) J. Biol. Chem. 251, 4398-4407], we found that reductive methylation with 2 mM formaldehyde (assembly blocked) did not decrease carbamate formation (carbamate formation was inhibited at higher formaldehyde concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)     

Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III

Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.     

Investigation of adenosinetriphosphatase activity of rat liver and thymus cell nuclei]

The activity of ATPase was studied in highly purified rat liver and thymus cell nuclei, HCO3-, CO3(2-) and SO3(2-) stimulated nuclear ATPase in 1.5--2 times. HSO3- did not affect the enzyme activity, and NO3-, J-, ClO4-,F- and SCN- inhibited it. Bicarbonate increased V and decreased Ka for ATP. SCN- inhibited HCO3--ATPase activity non-competitively with respect to HCO3-. Mg2+-ATPase activity did not depend on pH, and HCO3-component of the activity was decreased under alkaline pH. Mg2+, Mn2+ and Co2+ increased the initial ATPase activity and helped its stimulation with HCO3-. Ba2+, Ni2+ and Zn2+ inhibited the ATPase activity, and Ca2+ did not affect it, Nuclear ATPase is sensitive to 2,4-dinitrophenol and DNAase. It is suggested that cell nuclei have their own H+-ATPase differing for some characteristics from mitochondrial H+-ATPase.     

Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana

We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconversion in alpha-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A. thaliana beta-carbonic anhydrase provide direct evidence for the role of this residue in beta-carbonic anhydrase. Namely, the mutation of Gln-158 to Ala results in a significant decrease in the maximal k(cat) (33% of wild type) at steady state and the maximal rate of CO(2)(-) HCO(2)(-) exchange at chemical equilibrium as measured by R(1)/[E] (7% of wild type), while leaving the maximal rate of H(+) transfer, as measured by k(cat) at steady state, or R(H(2)O)) at chemical equilibrium, largely unaffected.     

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.     

Manganese-containing superoxide dismutase from Escherichia coli: reversible resolution and metal replacements.

Exposure of the manganese-containing Superoxide dismutase of Escherichia coli to pH 3.2, in the presence of 0.7 m guanidinium chloride, causes a rapid loss of manganese and of activity. The apoenzyme so produced can be reconstituted by addition of MnCl₂ followed by neutralization. In contrast, manganese cannot be restored to the apoenzyme by adding MnCl₂after neutralization. The reconstituted enzyme is indistinguishable from the native enzyme in terms of its catalytic activity or electrophoretic behavior on polyacrylamide gels. Co(II), Ni(II), Zn(II), Fe(II), or Cu(II) could compete with Mn(II) during reconstitution of the apoenzyme. In the cases of Co(II), Ni(II), and Zn(II), it was shown that, in preventing reconstitution by Mn(II), they were themselves bound to the enzyme in stoichiometric amounts, in place of Mn(II). The binding of Fe(II) was also explored and was distinct in that the enzyme could bind more than stoichiometric amounts of this metal. None of the derivatives, in which Mn(II) had been replaced by another metal, were catalytically active. Nevertheless, these derivatives could be again resolved by exposure to acid guanidinium chloride and could then be converted back into the active holoenzyme by neutralization after addition of MnCl₂. It appears that the active site of this enzyme can accommodate and can tightly bind several metals other than manganese, but exhibits activity only with manganese. It also appears that movement of metal out of or into this site is only feasible at low pH and in the presence of a chaotropic agent. A substantial amount of the cobalt-substituted enzyme was prepared and its optical properties were recorded.     

Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser-Lys dyad mechanism.

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.     

Roles of metal ions in the maintenance of the tertiary and quaternary structure of arginase from Saccharomyces cerevisiae

Arginase from Saccharomyces cerevisiae has long been known to be a metal ion-requiring enzyme as it requires heating at 45 degrees C in the presence of 10 mM Mn2+ for catalytic activation. Metals are also thought to play a structural role in the enzyme, but the identity of the structural metal and its precise structural role have not been defined. Analysis of the metal ions that bind to yeast arginase by atomic absorption spectroscopy reveals that there is a weakly associated Mn2+ that binds to the trimeric enzyme with a stoichiometry of 1.04 +/- 0.05 mol of Mn2+ bound per subunit and an apparent K'D value of 26 microM at pH 7.0 and 4 degrees C. A more tightly associated Zn2+ ion can only be removed by dialysis against chelating agents. In occasional preparations, this site contained some Mn2+; however, Zn2+ and Mn2+ together bind to high affinity sites with a stoichiometry of 1.14 +/- 0.25/mol of subunit. Both the loosely associated catalytic Mn2+ ion and the more tightly associated structural Zn2+ ion confer stability to the enzyme. Removal of the weakly bound Mn2+ ion results in a 3 degree C decrease in the midpoint of the thermal transition (T 1/2) (from 57 by 54 degrees C) as monitored by UV difference absorption spectroscopy. Removal of the tightly bound Zn2+ ion produces a 19 degrees C decrease in T 1/2 (to 38 degrees C). Similar results are obtained by circular dichroism measurements. When the Zn2+ ion is removed, the steady-state fluorescence intensity increases 100% as compared to the holoenzyme, with a shift in the emission maximum from 337 to 352 nm. This suggests that in the folded trimeric metalloenzyme, the tryptophan fluorescence is quenched and that upon removal of the structural metal, the quenching is relieved as tryptophan residues become exposed to more polar environments. Equilibrium sedimentation experiments performed after dialysis of the enzyme against EDTA demonstrate that arginase exists in a reversible monomer-trimer equilibrium, in the absence of metal ions, with a KD value of 5.05 x 10(-11) M2. In contrast, the native enzyme exists as a trimer with no evidence of dissociation when Mn2+ and Zn2+ are present (Eisenstein, E., Duong, L.T., Ornberg, R. L., Osborne, J.C., Jr., and Hensley, P. (1986) J. Biol. Chem. 261, 12814-12819). In summary, the study presented here demonstrates that binding of a weakly bound Mn2+ ion confers catalytic activity. In contrast, binding of a more tightly associated Zn2+ ion confers substantial stability to the tertiary and quaternary structure of the enzyme.     

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.     

The role of CO2 in cobalt-catalyzed peroxidations

Augmentation, by CO(2)/HCO(3)(-), of Co(II)-catalyzed peroxidations was explored to clarify whether the rate enhancement was due to CO(2) or to HCO(3)(-). The rate of oxidation of NADH by Co(II) plus H(2)O(2), in Tris or phosphate, was markedly enhanced by CO(2)/HCO(3)(-). Phosphate was seen to inhibit the Co(II)-catalyzed peroxidation, probably due to its sequestration of the Co(II). When CO(2) was used, there was an initial burst of NADH oxidation followed by a slower linear rate. The presence of carbonic anhydrase eliminated this initial burst; establishing that CO(2) rather than HCO(3)(-) was the species responsible for the observed rate enhancements. Both kinetic and spectral data indicated that Co(II) was converted by H(2)O(2) into a less active form from which Co(II) could be regenerated. This less active form absorbed in both the UV and visible regions, and is assumed to be a peroxy bridged binuclear complex. The rate of formation of this absorbing form was increased by HCO(3)(-)/CO(2). A minimal mechanism consistent with these observations is proposed.     

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.     

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.