Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).     

Association-dissociation of mammalian brain glutamine synthetase: effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn4E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982) Biochemistry 24, 6389-6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 micrograms/ml (20 nM octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn4EMn4 could be activated further by Mn(II) to form a species Mn4EMn4Mn8 that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn4EMn4, associated to octamers more extensively than Mn4E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and L-glutamine, or ATP and L-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a Kd value of ca. 2.5 X 10(-6) M, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 +/- 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 M urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (+/- 20) for tetramers in solution. Light-scattering experiments showed that, with 1.7-2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn4EMn4Mn8)n, where n greater than or equal to 5. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pseudomonas aeruginosa contains a novel type V porphobilinogen synthase with no required catalytic metal ions.

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I-IV) by the number of Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8-16 divalent ions. These four Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.     

Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta

The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.     

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.     

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

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

Oligomeric state of the muscle glycogen phosphorylase b in the system of reversed micelles of aerosol OT in octane

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscle was studied in the system of aerosol OT (AOT) reversed micelles in octane. The sedimentation experiments have shown that the enzyme oligomeric state depends on the degree of micelle hydration. The enzyme monomer, dimer, trimer, tetramer, hexamer, and octamer were observed, depending on the degree of hydration.     

The aggregation and dissociation properties of a low molecular weight mannose 6-phosphate receptor from bovine testis

The aggregation state of low molecular weight mannose 6-phosphate receptor from bovine testis was determined in membrane preparations and in purified soluble preparations. The effect of aggregation on binding of the receptor to immobilized pentamannose 6-phosphate was also examined. Nonreducing SDS-PAGE followed by immunoblotting revealed that interchain disulfide bonds exist in detergent-solubilized and purified receptor preparations, but not in membrane-associated receptor. Reduction of the receptor with dithiothreitol abolished its ligand binding activity and drastically altered its ability to bind antibodies. The results of receptor crosslinking and molecular sieving chromatography studies suggest that the receptor exists in membranes as a noncovalently linked dimer and in solution as oligomeric forms, largely as a tetramer. The formation of the tetramer is affected by the concentration of the receptor, but not by its solubilization from membranes with detergent, nor by the presence of mannose 6-phosphate. Mono-, di-, and tetramer forms of 125I-labeled receptor were separated by molecular sieving chromatography and examined for their ability to bind to immobilized ligand, agarose-pentamannose-phosphate. The order of binding observed was tetramer greater than dimer greater than monomer. Binding of the monomer and dimer to immobilized ligand was dependent on the presence of divalent cations while the tetramer had little requirement for divalent cations.     

The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ > Mn2+ > Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.     

Properties of creatine kinase from skeletal muscle mitochondria

Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.     

Association-dissociation of mammalian brain glutamine synthetase: Effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn₄E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982)Biochemistry24, 6389–6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 μg/ml (20 nm octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn₄EMn₄ could be activated further by Mn(II) to form a species Mn₄EMn₄Mn₈ that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn₄EMn₄, associated to octamers more extensively than Mn₄E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and l-glutamine, or ATP and l-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a K_d value of ca. 2.5 × 10^?6m, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 ± 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 m urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (±20) for tetramers in solution. Light-scattering experiments showed that, with 1.7–2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn₄EMn₄Mn₈)_n, where $\text{n}\text{?}\text{? 5}$. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer. Analysis of various stereochemical models for the interaction of subunit domains (assuming identical subunits) within tetramers, between tetramers in the octamers, and between octamers indicate that the data are most consistent with isologous, rather than heterologous, interactions to produce octamer. These analyses also predict that formation of oligomers from cubic octamers through weaker, Mn(II)-dependent interactions also are most likely to occur via isologous domains. The available electron micrographic evidence support these hypothetical models. Interactions within tetramers are stronger than those between tetramers, which are stronger than those between octamers.     

Metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase

Bovine lens leucyl aminopeptidase (blLAP), a homohexameric metallopeptidase preferring bulky and hydrophobic amino acids at the N-terminus of (di)peptides, contains two Zn(2+) ions per subunit that are essential for catalytic activity. They may be replaced by other divalent cations with different exchange kinetics. The protein readily exchangeable site (site 1) can be occupied by Zn(2+), Mn(2+), Mg(2+), or Co(2+), while the tight binding site (site 2) can be occupied by Zn(2+) or Co(2+). We recently reported that introduction of Mn(2+) into site 1 generates a novel activity of blLAP toward CysGly [Cappiello, M., et al. (2004) Biochem. J. 378, 35-44], which in contrast is not hydrolyzed by the (Zn/Zn) enzyme. This finding, while disclosing a potential specific role for blLAP in glutathione metabolism, raised a question about the features required for molecules to be a substrate for the enzyme. To clarify the interaction of the enzyme with sulfhydryl-containing derivatives, (Zn/Zn)- and (Mn/Zn)blLAP forms were prepared and functional-structural studies were undertaken. Thus, a kinetic analysis of various compounds with both enzyme forms was performed; the crystal structure of (Zn/Zn)blLAP in complex with the peptidomimetic derivative Zofenoprilat was determined, and a modeling study on the CysGly-(Zn/Zn)blLAP complex was carried out. This combined approach provided insight into the interaction of blLAP with sulfhydryl-containing derivatives, showing that the metal exchange in site 1 modulates binding to these molecules that may result in enzyme substrates or inhibitors, depending on the nature of the metal.     

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.     

Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese

All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg(2+) is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn(2+) in vitro. Here we report on the effects of Mn(2+) and Mg(2+) on the enzymatic properties of human DNA polymerase iota (pol iota). pol iota exhibited the greatest activity in the presence of low levels of Mn(2+) (0.05-0.25 mm). Peak activity in the presence of Mg(2+) was observed in the range of 0.1-0.5 mm and was significantly reduced at concentrations >2 mm. Steady-state kinetic analyses revealed that Mn(2+) increases the catalytic activity of pol iota by approximately 30-60,000-fold through a dramatic decrease in the K(m) value for nucleotide incorporation. Interestingly, whereas pol iota preferentially misinserts G opposite T by a factor of approximately 1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mm Mg(2+), respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mm Mn(2+). Low levels of Mn(2+) also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol iota for Mn(2+) even when Mg(2+) is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn(2+) rather than Mg(2+), as tacitly assumed.     

Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.     

Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys substitutions by Mn2+ in vitro

The D,DX(35)E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg(2+) or Mn(2+)), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg(2+) and Mn(2+), and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX(35)E motif. Mn(2+) but not Mg(2+) can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn(2+) only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex.     

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

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

Conditions for reciprocal conversion of oligomeric forms of heart mitochondrial creatine kinase

A procedure for purifying creatine kinase from bovine heart mitochondria, including enzyme extraction from mitochondria with salt solutions, concentration on cellulose phosphate gel and gel filtration on Sephacryl S-300 has been developed. Using ultracentrifugation in a sucrose density gradient and gel filtration, it was demonstrated that mitochondrial creatine kinase is present in solution as a mixture of two main forms, i. e., an octamer and a dimer. The distribution of the oligomeric forms is not influenced by changes in the ionic strength from 0.02 to 0.25, temperature (5-20 degrees C), freezing-thawing and the nature of monovalent anions (Cl-, NO3-, CH3COO-) and cations (Na+, K+) present in the medium. At pH 6.0, the predominant form is the octamer; an increase in pH induces its dissociation. An equilibrious mixture of the creatine kinase reaction substrates in the presence of Mg2+ also causes octamer dissociation; no dissociation is observed in the absence of Mg2+ or in the presence of one of the substrates. The non-working couple of substrates, Mg-ADP and creatine, causes dissociation of the octamer in the presence of Cl-, but not of CH3COO-. It is assumed that the dissociating effect of the substrates is due to conformational changes in the subunits concomitant with the formation of the creatine kinase active center in the course of catalysis. At physiological concentrations of nucleotide substrates, the degree of octamer dissociation depends on pH, creatine phosphate/creatine ratio and Pi. It is assumed that the above factors may regulate the reversible conversion of the octamer into the dimer in vivo.     

Zinc-dependent dimerization of the folding catalyst, protein disulfide isomerase

Protein disulfide isomerase (PDI), an essential folding catalyst and chaperone of the endoplasmic reticulum (ER), has four structural domains (a-b-b'-a'-) of approximately equal size. Each domain has sequence or structural homology with thioredoxin. Sedimentation equilibrium and velocity experiments show that PDI is an elongated monomer (axial ratio 5.7), suggesting that the four thioredoxin domains are extended. In the presence of physiological levels (<1 mM) of Zn(2+) and other thiophilic divalent cations such as Cd(2+) and Hg(2+), PDI forms a stable dimer that aggregates into much larger oligomeric forms with time. The dimer is also elongated (axial ratio 7.1). Oligomerization involves the interaction of Zn(2+) with the cysteines of PDI. PDI has active sites in the N-terminal (a) and C-terminal (a')thioredoxin domains, each with two cysteines (CGHC). Two other cysteines are found in one of the internal domains (b'). Cysteine to serine mutations show that Zn(2+)-dependent dimerization occurs predominantly by bridging an active site cysteine from either one of the active sites with one of the cysteines in the internal domain (b'). The dimer incorporates two atoms of Zn(2+) and exhibits 50% of the isomerase activity of PDI. At longer times and higher PDI concentrations, the dimer forms oligomers and aggregates of high molecular weight (>600 kDa). Because of a very high concentration of PDI in the ER, its interaction with divalent ions could play a role in regulating the effective concentration of these metal ions, protecting against metal toxicity, or affecting the activity of other (ER) proteins that use Zn(2+) as a cofactor.