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.     

Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii

Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn(2+) rather than Mg(2+), the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and II PPases. We measured rates of PP(i) hydrolysis, PP(i) synthesis, and P(i)/water oxygen exchange catalyzed by sgPPase with Mn(2+), Mg(2+), or Co(2+) in the high-affinity metal-binding site and Mg(2+) in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P(i)). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PP(i) complex isomerization known to occur in family I PPases; (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PP(i) hydrolysis and product release; (c) the marked acceleration of sgPPase catalysis in the presence of Mn(2+) and Co(2+) results from a combined effect of these ions on bound PP(i) hydrolysis and P(i) release; (d) sgPPase exhibits lower affinity for both PP(i) and P(i); and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K(m), which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PP(i) concentrations).     

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.     

Structural studies of metal ions in family II pyrophosphatases: the requirement for a Janus ion

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn(2+) and Co(2+) preferentially activate family II PPases; Mg(2+) partially activates; and Zn(2+) can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn(2+) at 1.3 A resolution, and, at 2.05 A resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn(2+). Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn(2+) or Co(2+), as an activator rather than Mg(2+). Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn(2+) and Co(2+), which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn(2+) activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205-Gln81-Gln80 form a hydrophilic channel to speed product release from the active site.     

Quaternary structure and metal ion requirement of family II pyrophosphatases from Bacillus subtilis, Streptococcus gordonii, and Streptococcus mutans

Pyrophosphatase (PPase) from Bacillus subtilis has recently been found to be the first example of a family II soluble PPase with a unique requirement for Mn2+. In the present work, we cloned and overexpressed in Escherichia coli putative genes for two more family II PPases (from Streptococcus mutans and Streptococcus gordonii), isolated the recombinant proteins, and showed them to be highly specific and active PPases (catalytic constants of 1700-3300 s(-)1 at 25 degrees C in comparison with 200-400 s(-)1 for family I). All three family II PPases were found to be dimeric manganese metalloenzymes, dissociating into much less active monomers upon removal of Mn2+. The dimers were found to have one high affinity manganese-specific site (K(d) of 0.2-3 nm for Mn2+ and 10-80 microm for Mg2+) and two or three moderate affinity sites (K(d) approximately 1 mm for both cations) per subunit. Mn2+ binding to the high affinity site, which occurs with a half-time of less than 10 s at 1.5 mm Mn2+, dramatically shifts the monomer <--> dimer equilibrium in the direction of the dimer, further activates the dimer, and allows substantial activity (60-180 s(-)1) against calcium pyrophosphate, a potent inhibitor of family I PPases.     

Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers.

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.     

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.     

Probing essential water in yeast pyrophosphatase by directed mutagenesis and fluoride inhibition measurements

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.     

The electrophilic and leaving group phosphates in the catalytic mechanism of yeast pyrophosphatase

Binding of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase) active site occurs at two subsites, P1 and P2. Mutations at P2 subsite residues (Y93F and K56R) caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn(2+) or Co(2+) than mutations at P1 subsite residues (R78K and K193R). Phosphate binding was estimated in these experiments from the inhibition of ATP hydrolysis at a sub-K(m) concentration of ATP. Tight phosphate binding required four Mn(2+) ions/active site. These data identify P2 as the high affinity subsite and P1 as the low affinity subsite, the difference in the affinities being at least 250-fold. The time course of five "isotopomers" of phosphate that have from zero to four (18)O during [(18)O]P(i)-[(16)O]H(2)O oxygen exchange indicated that the phosphate containing added water is released after the leaving group phosphate during pyrophosphate hydrolysis. These findings provide support for the structure-based mechanism in which pyrophosphate hydrolysis involves water attack on the phosphorus atom located at the P2 subsite of PPase.     

Binding of substrate at the effector site of pyrophosphatase increases the rate of its hydrolysis at the active site

It is shown that in addition to the active site, each subunit of Escherichia coli inorganic pyrophosphatase (E-PPase) contains an extra binding site for the substrate magnesium pyrophosphate or its non-hydrolyzable analog magnesium methylenediphosphonate. The occupancy of the extra site stimulates the substrate conversion. Binding affinity of this site decreased or disappeared upon the conversion of E-PPase into a trimeric form or introduction of point mutations. However, when the slowly hydrolyzed substrate, lanthanum pyrophosphate (LaPP(i)), is used, the extra site was revealed in all enzyme forms of E-PPase and of Y-PPase (Saccharomyces cerevisiae PPase), resulting in about 100-fold activation of hydrolysis. A hypothesis on the localization of the extra site and the mechanism of its effect in E-PPase is presented.     

Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase.

The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 A. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 A. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion. The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 A/Calpha. But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.     

An unusual, His-dependent family I pyrophosphatase from Mycobacterium tuberculosis

Soluble inorganic pyrophosphatases (PPases) comprise two evolutionarily unrelated families (I and II). These two families have different specificities for metal cofactors, which is thought to be because of the fact that family II PPases have three active site histidines, whereas family I PPases have none. Here, we report the structural and functional characterization of a unique family I PPase from Mycobacterium tuberculosis (mtPPase) that has two His residues (His21 and His86) in the active site. The 1.3-A three-dimensional structure of mtPPase shows that His86 directly interacts with bound sulfate, which mimics the product phosphate. Otherwise, mtPPase is structurally very similar to the well studied family I hexameric PPase from Escherichia coli, although mtPPase lacks the intersubunit metal binding site found in E. coli PPase. The cofactor specificity of mtPPase resembles that of E. coli PPase in that it has high activity in the presence of Mg2+, but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn2+ or Zn2+. Replacements of His21 and His86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg2+- and Mn2+-supported reactions (H86K) or reduced Mg2+-supported activity (H21K). However, both replacements markedly increased the Zn2+-supported activity of mtPPase (up to 11-fold). In the double mutant, Zn2+ was a 2.5-fold better cofactor than Mg2+. These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity.     

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.     

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.     

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.     

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.     

Characterization of nuclear phosphoprotein phosphatases from goat testis

Two nuclear phosphoprotein phosphatases (PPases I and II) that cause dephosphorylation of [32P]histone, have been partially purified from goat testis. The enzymic activity is associated with nucleoplasm and chromatin. PPase I is markedly stimulated (approx. 200-600%) by Mg2+ or Mn2+ (1 mM) whereas Ca2+ (1 mM) causes slight stimulation (approx. 35%) of the enzyme. On the contrary, PPase II is only slightly activated (20-40%) by these metal ions (5 mM). Both the phosphoprotein phosphatase isoenzymes are maximally active at pH 6-7. PPases I and II are strongly inhibited (approx. 60-100%) by ZnCl2 (1 mM), P1 (5 mM) and thiol reagents. NaF (5 mM) inhibits (approx. 40%) specifically the activity of PPase I rather than PPase II. PPases are strongly inhibited by relatively high concentration of NaCl (0.4 M), isoenzyme II being more sensitive (approx. 80%) than isoenzyme I (approx. 50%). In addition to histones, both the isoenzymes can as well cause dephosphorylation of protamine, casein, and testicular nuclear proteins. Enzymic characteristics of the testicular nuclear PPases are clearly different from those of the cytosolic enzyme previously characterized.     

Effect of the Met344His mutation on the conformational dynamics of bovine beta-1,4-galactosyltransferase: crystal structure of the Met344His mutant in complex with chitobiose

Beta-1,4-galactosyltransferase (beta4Gal-T1) in the presence of manganese ion transfers galactose from UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) that is either free or linked to an oligosaccharide. Crystallographic studies on bovine beta4Gal-T1 have shown that the primary metal binding site is located in the hinge region of a long flexible loop, which upon Mn(2+) and UDP-Gal binding changes from an open to a closed conformation. This conformational change creates an oligosaccharide binding site in the enzyme. Neither UDP nor UDP analogues efficiently induce these conformational changes in the wild-type enzyme, thereby restricting the structural analysis of the acceptor binding site. The binding of Mn(2+) involves an uncommon coordination to the Sdelta atom of Met344; when it is mutated to His, the mutant M344H, in the presence of Mn(2+) and UDP-hexanolamine, readily changes to a closed conformation, facilitating the structural analysis of the enzyme bound with an oligosaccharide acceptor. Although the mutant M344H loses 98% of its Mn(2+)-dependent activity, it exhibits 25% of its activity in the presence of Mg(2+). The crystal structures of M344H-Gal-T1 in complex with either UDP-Gal.Mn(2+) or UDP-Gal.Mg(2+), determined at 2.3 A resolution, show that the mutant enzyme in these complexes is in a closed conformation, and the coordination stereochemistry of Mg(2+) is quite similar to that of Mn(2+). Although either Mn(2+) or Mg(2+), together with UDP-Gal, binds and changes the conformation of the M344H mutant to the closed one, it is the Mg(2+) complex that engages efficiently in catalyses. Thus, this property enabled us to crystallize the M344H mutant for the first time with the acceptor substrate chitobiose in the presence of UDP-hexanolamine and Mn(2+). The crystal structure determined at 2.3 A resolution reveals that the GlcNAc residue at the nonreducing end of chitobiose makes extensive hydrophobic interactions with the highly conserved Tyr286 residue.     

Effectory site in Escherichia coli inorganic pyrophosphatase is revealed upon mutation at the intertrimeric interface

Escherichia coli inorganic pyrophosphatase (E-PPase) is a homohexamer formed from two trimers related by a two-fold axis. The residue Asp26 participates in intertrimeric contacts. Kinetics of MgPPi hydrolysis by a mutant Asp26Ala E-PPase is found to not obey Michaelis-Menten equation but can be described within the scheme of activation of hydrolysis by a free PPi binding at an effectory subsite. Existence of such a subsite is confirmed by the finding that the free form of methylenediphosphonate activates MgPPi hydrolysis though its magnesium complex is a competitive inhibitor. The Asp26Ala variant is the first example of hexameric E-PPase demonstrated to have an activatory subsite.     

The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning.

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition.