Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies. Alteration of substrate specificity by replacement of Asn149 with Asp

To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The K(m) for arginine (1.4-1.6 mM) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (K(m) = 2.5 +/- 0.2 mM) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn(2+) and contained < 0.1 Mn(2+).subunit(-1), whereas wild-type and His145Asn enzymes were half active and contained 1.1 +/- 0.1 Mn(2+).subunit(-1) and 1.3 +/- 0.1 Mn(2+).subunit(-1), respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with K(d) of 1.8 +/- 0.2 x 10(-8) M for the wild-type and His145Asn enzymes and 16.2 +/- 0.5 x 10(-8) m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (lambda(max) = 338-339 nm) and sensitivity to thermal inactivation were not altered. The Asn149-->Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn(2+) (A) is the more tightly bound metal ion in arginase II.     

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.     

Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants

The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined. About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively. Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme. However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer. We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant.     

Mg2+ is bound to glutamine synthetase extracted from bovine or ovine brain in the presence of L-methionine-S-sulfoximine phosphate

Purified glutamine synthetase from bovine or ovine brain had no tightly bound Mn2+. By extraction of bovine or ovine brain glutamine synthetase in the presence of L-Met-S-sulfoximine phosphate and ADP in metal ion-free water and 0.1 M KCl, only endogenously bound divalent cations were trapped on the enzyme. Enzyme complexes isolated by immunoprecipitation contained less than 0.05 Mn2+ and 1.5 +/- 0.2 Mg2+ per subunit. Without inactive complex formation, the enzyme immunoprecipitated from extracts contained undetectable Mn2+ (less than 0.01 eq per subunit) and 0.1-2.0 eq of Mg2+ per subunit. Direct binding measurements showed that the purified bovine brain enzyme contained two divalent cations bound at the active site of each subunit. Thus, although either Mg2+ or Mn2+ supports enzyme activity in vitro, Mg2+ rather than Mn2+ appears to be bound to brain glutamine synthetase in vivo.     

Structure and catalytic properties of an engineered heterodimer of enolase composed of one active and one inactive subunit

Enolase is a dimeric enzyme that catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. This reversible dehydration is effected by general acid-base catalysis that involves, principally, Lys345 and Glu211 (numbering system of enolase 1 from yeast). The crystal structure of the inactive E211Q enolase shows that the protein is properly folded. However, K345 variants have, thus far, failed to crystallize. This problem was solved by crystallization of an engineered heterodimer of enolase. The heterodimer was composed of an inactive subunit that has a K345A mutation and an active subunit that has N80D and N126D surface mutations to facilitate ion-exchange chromatographic separation of the three dimeric species. The structure of this heterodimeric variant, in complex with substrate/product, was obtained at 1.85 A resolution. The structure was compared to a new structure of wild-type enolase obtained from crystals belonging to the same space group. Asymmetric dimers having one subunit exhibiting two of the three active site loops in an open conformation and the other in a conformation having all three loops closed appear in both structures. The K345A subunit of the heterodimer is in the loop-closed conformation; its Calpha carbon atoms closely match those of the corresponding subunit of wild-type enolase (root-mean-squared deviation of 0.23 A). The kcat and kcat/Km values of the heterodimer are approximately half those of the N80D/N126D homodimer, which suggests that the subunits in solution are kinetically independent. A comparison of enolase structures obtained from crystals belonging to different space groups suggests that asymmetric dimers can be a consequence of the asymmetric positioning of the subunits within the crystal lattice.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Exploring the catalytic center of TaqI endonuclease: rescuing catalytic activity by double mutations and Mn2+

TaqI is a metal-dependent endonuclease that recognizes T(downward arrow)CGA, with the arrow indicating the cleavage site. Mutations at K158 render the enzyme inactive and mutations at K157 significantly reduce DNA cleavage activity (W. Cao and F. Barany (1998) J. Biol. Chem. 273, 33002-33010). Aspartate, glutamate, and histidine substitutions were made at K158 in the wild-type and K157S mutant TaqI endonuclease to understand the functional organization of the active site. None of the mutants was active with Mg(2+), but the DNA cleavage activities were partly rescued by Mn2+ for K157S-K158E and K157S-K158H mutants. The rescuing effects were observed with Mn2+ but not with other divalent cations. K157S-K158E required higher Mn2+ concentrations than the wild-type enzyme for DNA cleavage activity, suggesting that a Mn2+ ion is weakly bound at the 158 position. The need to neutralize K157 to recover the catalytic activity of K158E and K158H indicates that K158 and K157 may interact functionally. In analogy with EcoRV, Ca2+ stimulated Mn2+-mediated cleavage for the wild-type TaqI, suggesting the existence of at least two metal ions at the catalytic center. A catalytic mechanism involving two metal ions and the K157-K158 pair is proposed for TaqI endonuclease.     

Histidine 268 in 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase plays the same role as histidine 202 in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (Phe) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first order with respect to enzyme and DEPC concentrations with a pseudo-second order rate constant of inactivation by DEPC of 4.9 +/- 0.8 m(-1) s(-1) at pH 6.8 and 4 degrees C. The dependence of inactivation on pH and the spectral features of enzyme modified at specific pH values imply that both histidine and cysteine residues are modified, which is confirmed by site-directed mutagenesis. Analysis of the chemical modification data indicates that one histidine is essential for activity. DAH 7-P synthase (Phe) is protected against DEPC inactivation by phosphoenolpyruvate, whereas d-erythrose 4-phosphate offers only minimal protection. The conserved residues H-172, H-207, H-268, and H-304 were individually mutated to glycine. The H304G and H207G mutants retain some level of activity, whereas the H268G and H172G mutants are virtually inactive. A comparison of the circular dichroism spectra of wild-type enzyme and the various mutants demonstrates that H-172 may play a structural role. Comparison of the UV spectra of the H268G and wild-type enzymes saturated with Cu(2+) indicates that the metal-binding site of the H268G mutant resembles that of the wild-type enzyme. The residue H-268 may play a catalytic role based on the site-directed mutagenesis and spectroscopic studies. Cysteine 61 appears to influence the pK(a) of H-268 in the wild-type enzyme. The pK(a) of H-268 increases from 6.0 to 7.0 following mutation of C-61 to glycine.     

Activation and inhibition of rubber transferases by metal cofactors and pyrophosphate substrates

Metal cofactors are necessary for the activity of alkylation by prenyl transfer in enzyme-catalyzed reactions. Rubber transferase (RuT, a cis-prenyl transferase) associated with purified rubber particles from Hevea brasiliensis, Parthenium argentatum and Ficus elastica can use magnesium and manganese interchangably to achieve maximum velocity. We define the concentration of activator required for maximum velocity as [A](max). The [A](max)(Mg2+) in F. elastica (100 mM) is 10 times the [A](max)(Mg2+) for either H. brasiliensis (10 mM) or P. argentatum (8 mM). The [A](max)(Mn2+) in F. elastica (11 mM), H. brasiliensis (3.8 mM) and P. argentatum (6.8 mM) and the [A](max)(Mg2+) in H. brasiliensis (10 mM) and P. argentatum (8 mM) are similar. The differences in [A](max)(Mg2+) correlate with the actual endogenous Mg(2+) concentrations in the latex of living plants. Extremely low Mn(2+) levels in vivo indicate that Mg(2+) is the RuT cofactor in living H. brasiliensis and F. elastica trees. Kinetic analyses demonstrate that FPP-Mg(2+) and FPP-Mn(2+) are active substrates for rubber molecule initiation, although free FPP and metal cations, Mg(2+) and Mn(2+), can interact independently at the active site with the following relative dissociation constants K(d)(FPP) 相似文献   

Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2. Activation by sodium and absolute dependence on bivalent cations

We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.     

Non-chelating inhibition of the H101N variant of human liver arginase by EDTA

Recombinant wild-type human liver arginase (EC 3.5.3.1) expressed in Escherichia coli was markedly resistant to inhibition by ethylene diamine tetraacetic acid (EDTA). In contrast, half and fully activated species of the H101N variant were totally inactive in the presence of approximately 1 mM EDTA. Dilution of inhibited species in metal-free buffer lead to a time dependent recovery of activity, even when measured in the absence of added Mn2+. The inhibition was mixed type, with predominance of a competitive component (Kii=0.31 mM; Kis=0.022 mM). The structurally related N,N,N',N'-tetramethylethylenediamine was not inhibitory, indicating the importance of the carboxyl groups in EDTA inhibition. We conclude that EDTA inhibition of H101N arginase is not due to interaction with a weakly bound Mn2+ or chelation of essential metal ions.     

Subunit a of the yeast V-ATPase participates in binding of bafilomycin

Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H(+))-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V(0) domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed K(i) values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nm). These included E721K (K(i) 0.38 +/- 0.03 nm), L724A (0.40 +/- 0.02 nm), and N725F (0.54 +/- 0.06 nm). Only the N725F mutation displayed a K(i) for concanamycin (0.84 +/- 0.04 nm) that was slightly higher than wild-type (0.60 +/- 0.07 nm). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.     

Role of metal ions in the reaction catalyzed by L-ribulose-5-phosphate 4-epimerase

H97N, H95N, and Y229F mutants of L-ribulose-5-phosphate 4-epimerase had 10, 1, and 0.1%, respectively, of the activity of the wild-type (WT) enzyme when activated by Zn(2+), the physiological activator. Co(2+) and Mn(2+) replaced Zn(2+) in Y229F and WT enzymes, although less effectively with the His mutants, while Mg(2+) was a poorly bound, weak activator. None of the other eight tyrosines mutated to phenylalanine caused a major loss of activity. The near-UV CD spectra of all enzymes were nearly identical in the absence of metal ions and substrate, and addition of substrate without metal ion showed no effect. When both substrate and Zn(2+) were present, however, the positive band at 266 nm increased while the negative one at 290 nm decreased in ellipticity. The changes for the WT and Y229F enzymes were greater than for the two His mutants. With Co(2+) as the metal ion, the CD and absorption spectra in the visible region were different, showing little ellipticity in the absence of substrate and a weak absorption band at 508 nm. With substrate present, however, an intense absorption band at 555 nm (epsilon = 150-175) with a negative molar ellipticity approaching 2000 deg cm(2) dmol(-1) appears with WT and Y229F enzymes. With the His mutants, the changes induced by substrate were smaller, with negative ellipticity only half as great. The WT, Y229F, H95N, and H97N enzymes all catalyze a slow aldol condensation of dihydroxyacetone and glycolaldehyde phosphate with an initial k(cat) of 1.6 x 10(-3) s(-1). The initial rate slowed most rapidly with WT and H97N enzymes, which have the highest affinity for the ketopentose phosphates formed in the condensation. The EPR spectrum of enzyme with Mn(2+) exhibited a drastic decrease upon substrate addition, and by using H(2)(17)O, it was determined that there were three waters in the coordination sphere of Mn(2+) in the absence of substrate. These data suggest that (1) the substrate coordinates to the enzyme-bound metal ion, (2) His95 and His97 are likely metal ion ligands, and (3) Tyr229 is not a metal ion ligand, but may play another role in catalysis, possibly as an acid-base catalyst.     

Reaction of Ascaris suum phosphofructokinase with diethylpyrocarbonate. Inactivation and desensitization to allosteric modulation

Reaction of the phosphofructokinase from Ascaris suum with the reagent, diethylpyrocarbonate (DEPC), results in the loss of enzymatic activity. Treatment of the inactivated enzyme with hydroxylamine brings about the recovery of almost 80% of the original activity suggesting that the modified residues are histidines. Further evidence for the modification of histidines is that concomitant with the loss of activity, there is a change in A242 nm that corresponds to the derivatization of 5-6 histidines per subunit. There is no change in A278 nm during the derivatization process, thereby ruling out the modification of tyrosines by DEPC. Analyses of the first order inactivation rate constant for DEPC derivatization at different pH values resulted in the determination of a pKa of 6.4 +/- 0.1 for the group on the enzyme that reacts with DEPC. Derivatization of the enzyme with DEPC in the presence of fructose 6-phosphate (Fru-6-P) protected the enzyme against inactivation by 80%. ATP or MgATP gave no protection against DEPC inactivation. When the Fru-6-P-protected enzyme was further reacted with DEPC in the absence of Fru-6-P, a total of 2 histidines were modified per subunit, and the derivatization of one of these could be correlated with activity loss. When the phosphofructokinase that had been derivatized by DEPC in the presence of Fru-6-P was assayed, it was found that it no longer exhibited allosteric properties and appeared to be desensitized to ATP inhibition. This loss of ATP inhibition could be correlated with the modification of 2 histidines per subunit by DEPC. The first order rate constant for desensitization was determined at different pH values and a pKa value of 7.0 +/- 0.2 was obtained for the group(s) responsible for the desensitization. Regulatory studies with the desensitized enzyme revealed that the enzyme was not stimulated by AMP, NH4+, K+, phosphate, sulfate, or hexose bisphosphates. It is concluded that histidine may be involved both in the active site and the ATP inhibitory site of the ascarid phosphofructokinase.     

A molecular design that stabilizes active state in bacterial allosteric L-lactate dehydrogenases

l-Lactate dehydrogenase (l-LDH) of Lactobacillus casei (LCLDH) is a typical bacterial allosteric l-LDH that requires fructose 1,6-bisphosphate (FBP) for its enzyme activity. A mutant LCLDH was designed to introduce an inter-subunit salt bridge network at the Q-axis subunit interface, mimicking Lactobacillus pentosus non-allosteric l-LDH (LPLDH). The mutant LCLDH exhibited high catalytic activity with hyperbolic pyruvate saturation curves independently of FBP, and virtually the equivalent K(m) and V(m) values at pH 5.0 to those of the fully activated wild-type enzyme with FBP, although the K(m) value was slightly improved with FBP or Mn(2+) at pH 7.0. The mutant enzyme exhibited a markedly higher apparent denaturating temperature (T(1/2)) than the wild-type enzyme in the presence of FBP, but showed an even lower T(1/2) without FBP, where it exhibited higher activation enthalpy of inactivation (ΔH(?)). This result is consistent with the fact that the active state is more unstable than the inactive state in allosteric equilibrium of LCLDH. The LPLDH-like network appears to be conserved in many bacterial non-allosteric l-LDHs and dimeric l-malate dehydrogenases, and thus to be a key for the functional divergence of bacterial l-LDHs during evolution.     

W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition

Ca(+)-calmodulin (Ca(2+)-CaM)-dependent protein kinase II (Ca(2+)/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca(2+)-occupied CaM, and KN-93, an inhibitor of Ca(2+)/CaMKII, on the K(v)4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC(50) of 92.4 muM. The block was voltage dependent, with an effective electrical distance of 0.18 +/- 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of K(v)4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 muM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated K(v)4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 muM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca(2+)/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca(2+)-CaM, as well as a change in activity of Ca(2+)-CaM-dependent enzymes, including Ca(2+)/CaMKII.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta

Phosphotriesterase, isolated from the soil-dwelling bacterium Pseudomonas diminuta, catalyzes the detoxification of organophosphate-based insecticides and chemical warfare agents. The enzyme has attracted significant research attention in light of its possible employment as a bioremediation tool. As naturally isolated, the enzyme is dimeric. Each subunit contains a binuclear zinc center that is situated at the C-terminal portion of a "TIM" barrel motif. The two zincs are separated by approximately 3.4 A and coordinated to the protein via the side chains of His 55, His 57, His 201, His 230, Asp 301, and a carboxylated Lys 169. Both Lys 169 and a water molecule (or hydroxide ion) serve to bridge the two zinc ions together. Interestingly, these metals can be replaced with cadmium or manganese ions without loss of enzymatic activity. Here we describe the three-dimensional structures of the Zn(2+)/Zn(2+)-, Zn(2+)/Cd(2+)-, Cd(2+)/Cd(2+)-, and Mn(2+)/Mn(2+)-substituted forms of phosphotriesterase determined and refined to a nominal resolution of 1.3 A. In each case, the more buried metal ion, referred to as the alpha-metal, is surrounded by ligands in a trigonal bipyramidal ligation sphere. For the more solvent-exposed or beta-metal ion, however, the observed coordination spheres are either octahedral (in the Cd(2+)/Cd(2+)-, Mn(2+)/Mn(2+)-, and the mixed Zn(2+)/Cd(2+)-species) or trigonal bipyramidal (in the Zn(2+)/Zn(2+)-protein). By measuring the anomalous X-ray data from crystals of the Zn(2+)/Cd(2+)-species, it has been possible to determine that the alpha-metal ion is zinc and the beta-site is occupied by cadmium.     

Expression and characterization of the catalytic domains of soluble guanylate cyclase: interaction with the heme domain

The catalytic domains (alpha(cat) and beta(cat)) of alpha1beta1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. Alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn(2+) as compared to the physiological cofactor Mg(2+), although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K(M) for Mn(2+)-GTP was measured to be 85 +/- 18 microM, and in the presence of Mn(2+)-GTP, the K(D) for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.     

A PP2A active site mutant impedes growth and causes misregulation of native catalytic subunit expression

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the alpha4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression.