Biomedical subjects

R Lahti

Publications and source records attributed to R Lahti.

At least 37 records · Page 2Linked to original sources

PubMed1998

The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications.

We have solved the structure of two active-site variants of soluble inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85 and 2.15 A and R-factors of 19.5% and 18.3%, respectively. In the R78K variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction between P1 and Arg78, and solution data showing a decrease in P1 affinity in the variant. The structure explains why the mutation affects P1 and pyrophosphate binding much more than would be expected by the loss of one hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an interaction with P1. The R78K variant also provides the first direct evidence that the low-affinity phosphate group (P2) can adopt the structure that we believe is the immediate product of hydrolysis, with one of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase is, indeed, the attacking nucleophile. The D117E variant structure likewise supports our model of catalysis, as the Glu117 variant carboxylate group is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile is replaced by a carboxylate co-ordinated to two metal ions. Alternative confirmations of Glu117 may allow Wat1 to be present but at much reduced occupancy, explaining why the pKa of the nucleophile increases by three pH units, even though there is relatively little distortion of the active site. These new structures, together with parallel functional studies measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is considered unimportant for hydrolysis. They thus support the notion that PPase shares mechanistic similarity with the "two-metal ion" mechanism of polymerases.

Amino Acid Sequence↗

PubMed1998

Cloning and expression of a unique inorganic pyrophosphatase from Bacillus subtilis: evidence for a new family of enzymes.

An open reading frame located in the COTF-TETB intergenic region of Bacillus subtilis was cloned and expressed in Escherichia coli and shown to encode inorganic pyrophosphatase (PPase). The isolated enzyme is Mn2+-activated, like the authentic PPase isolated from B. subtilis. Although 13 functionally important active site residues are conserved in all 31 soluble PPase sequences so far identified, only two of them are conserved in B. subtilis PPase. This suggests that B. subtilis PPase represents a new family of soluble PPases (a Bs family), putative members of which were found in Archaeoglobus fulgidus, Methanococcus jannaschii, Streptococcus mutans and Streptococcus gordonii.

Amino Acid Sequence↗

PubMed1998

Evolutionary conservation of enzymatic catalysis: quantitative comparison of the effects of mutation of aligned residues in Saccharomyces cerevisiae and Escherichia coli inorganic pyrophosphatases on enzymatic activity.

Soluble inorganic pyrophosphatase (PPase) is one of the better understood phosphoryl-transfer enzymes and is distinctive in having four divalent metal ions at the active site. Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its active site variants and consider the effects of active site mutation on the pH-independent parameters and acid dissociation constants that characterize these profiles against the framework of the proposed structure of the activated complex. The results obtained (a) support the current mechanistic model in which a hydroxide ion, stabilized by binding to two metal ions at the active site and by an extended system of hydrogen bonds within the active site, is the nucleophile that attacks enzyme-bound inorganic pyrophosphate and (b) provide evidence that the acid group that is necessary for maximal activity is a water molecule coordinated to a third metal ion, as shown by the general rise in the pKa of this group that is a consequence of almost all of the mutations. We further compare the present results to those previously observed for the corresponding mutations in Escherichia coli PPase [E-PPase; Salminen et al. (1995) Biochemistry 34, 782-791]. Such comparison provides a measure of the extent to which different portions of the active site are conserved. We find that some corresponding mutations have different effects on catalytic function, demonstrating that even in the context of very similar active sites, interactions of the mutated site with less well conserved portions of the enzyme, in this case outside the active site, can lead to different outcomes. On the other hand, one region of the active site is highly conserved, suggesting that it may represent a common feature of phosphoryl-transfer enzymes or a vestige of a primitive ur-PPase active site.

Amino Acid Sequence↗

PubMed1998

Trimeric inorganic pyrophosphatase of Escherichia coli obtained by directed mutagenesis.

Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits. Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements of Mg2+ binding to trimer by equilibrium dialysis. The results of such measurements, together with the results of activity measurements as a function of [Mg2+] and pH, indicate that Mg2+ binds more weakly to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface. The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate. The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer. Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation. The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.

Enzyme Stability↗

PubMed1997

Structural and functional consequences of substitutions at the tyrosine 55-lysine 104 hydrogen bond in Escherichia coli inorganic pyrophosphatase.

Tyrosine 55 and lysine 104 are evolutionarily conserved residues that form a hydrogen bond in the active site of Escherichia coli inorganic pyrophosphatase (E-PPase). Here we used site-directed mutagenesis to examine their roles in structure stabilization and catalysis. Though these residues are not part of the subunit interface, Y55F and K104R (but not K104I) substitutions markedly destabilize the hexameric structure, allowing dissociation into active trimers on dilution. A K104I variant is nearly inactive while Y55F and K104R variants exhibit appreciable activity and require greater concentrations of Mg2+ and higher pH for maximal activity. The effects on activity are explained by (a) increased pK(a)s for the catalytically essential base and acid at the active site, (b) decreases in the rate constant for substrate (dimagnesium pyrophosphate) binding to enzyme-Mg2 complex vs enzyme-Mg3 complex, and (c) parallel decreases in the catalytic constant for the resulting enzyme-Mg2-substrate and enzyme-Mg3-substrate complexes. The results are consistent with the major structural roles of Tyr55 and Lys104 in the active site. The microscopic rate constant for PPi hydrolysis on either the Y55F or K104R variants increases, by a factor of 3-4 in the pH range 7.2-8.0, supporting the hypothesis that this reaction step depends on an essential base within the enzyme active site.

Biopolymers↗

PubMed1997

Effect of Asp-97-->Glu substitution on the pH dependence of catalysis by inorganic pyrophosphatase of Escherichia coli.

Substitution of Glu for the evolutionarily conserved Asp-97 in the active site of Escherichia coli inorganic pyrophosphatase increases the apparent pKa of the essential acidic group controlling the catalytic constant for pyrophosphate hydrolysis. In combination with previously reported data, this fact suggests that activity decreases in alkaline medium because of decreased rate of the release of the first molecule of the product phosphate from the active site.

Amino Acid Substitution↗

PubMed1996

The structural basis for pyrophosphatase catalysis.

BACKGROUND: Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable. RESULTS: We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 A resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions. CONCLUSIONS: Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.

Alkaline Phosphatase↗

PubMed1996

A site-directed mutagenesis study of Saccharomyces cerevisiae pyrophosphatase. Functional conservation of the active site of soluble inorganic pyrophosphatases.

We report the expression and initial characterization of 19 active-site variants of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase), including measurements of thermostability, oligomeric structure and specific activity at pH 7.2. 13 of the 19 conservative substitutions resulted in at least a fivefold decrease in activity, indicating that these residues are important for yeast PPase catalysis. The E58D, D117E, D120E and D152E variants had no activity under the conditions tested, suggesting that Glu58, Asp117, Asp120 and Asp152 may have crucial roles in catalysis. The effects of the mutations on catalytic activity were very similar to those observed with the corresponding variants of Escherichia coli PPase, proving conclusively that the active site and mechanism of soluble PPases are conserved. The D71E variant was more thermostable and the K56R, R78K, D115E and K154R variants were more thermolabile than the wild-type enzyme, whereas subunit:subunit interactions were somewhat weakened by the K56R, R78K, Y89F and K154R substitutions. These results suggest that Lys56, Asp71, Arg78, Tyr89, Asp115 and Lys154 are structurally important for yeast PPase.

Binding Sites↗

PubMed1996

Structure of Escherichia coli inorganic pyrophosphatase at 2.2 A resolution.

The refined crystal structures of hexameric soluble inorganic pyrophosphatase from E. coli (E-PPase) are reported to R factors of 18.7 and 18.3% at 2.15 and 2.2 A, respectively. The first contains one independent monomer; the other, two independent monomers, in an R32 unit cell. Because the E-PPase monomer is small with a large open active site, there are relatively few hydrophobic interactions that connect the active-site loops to the five-stranded twisted beta-barrel that is the hydrophobic core of the molecule. The active-site loops are, however, held in place by interactions between monomers around the threefold and twofold symmetry axes of the D(3) hexamer. Consequently, mutations of active-site residues (such as Glu20 and Lysl04) often affect protein stability and oligomeric structure. Conversely, mutations of residues in the interface between monomers (such as His136 and Hisl40) not only affect oligomeric structure but also affect active-site function. The effects of the H136Q and H140Q variants can be explained by the extended ionic interaction between H140, D143 and H136' of the neighbouring monomer. This interaction is further buttressed by an extensive hydrogen-bonding network that appears to explain why the E-PPase hexamer is so stable and also why the H136Q and H140Q variant proteins are less stable as hexamers.

Journal Article↗

PubMed1996

Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence.

Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM. The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised .

Catalysis↗

PubMed1996

Effect of E20D substitution in the active site of Escherichia coli inorganic pyrophosphatase on its quaternary structure and catalytic properties.

Glutamic acid 20 is an evolutionarily conserved residue found within the active site of the inorganic pyrophosphatase of Escherichia coli (E-PPase). Here we determine the effect of E20D substitution on the quaternary structure and catalytic properties of E-PPase. In contrast to wild-type enzyme, which is hexameric under a variety of conditions, E20D-PPase can be dissociated by dilution into nearly inactive trimers, as shown by electrophoresis of cross-linked enzyme, analytical ultracentrifugation, and measurement of catalytic activity as a function of enzyme concentration. Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply as the pH is lowered or raised from this value. Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases activity towards inorganic pyrophosphate (PPi) hydrolysis and oxygen exchange between water and inorganic phosphate (P1), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PPi in equilibrium with Pi in solution. Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D variant show that its decreased activity is largely accounted for on the basis of an increased pKa of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme-substrate complex, similar to what is seen for the D97E variant. By contrast, wild-type PPase catalysis over a wide range of Mg2+ concentration and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions. These results are consistent with a supporting role for Glu20 in PPase catalysis and demostrate that even conservative mutation at the active site can perturb the quaternary structure of the enzyme.

Binding Sites↗

PubMed1996

Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase.

We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively. Both structures contain two independent monomers in the asymmetric unit of an R32 cell. The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer. One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102. The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers. The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them. Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface. Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion. Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects. The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions. Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion.

Amino Acids↗

PubMed1996

An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Amino Acid Sequence↗

PubMed1995

Dissociation of hexameric Escherichia coli inorganic pyrophosphatase into trimers on His-136-->Gln or His-140-->Gln substitution and its effect on enzyme catalytic properties.

Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by glutamine. Significant changes in protein structure and activity were observed in the H136Q and H140Q variants only. In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by analytical ultracentrifugation and cross-linking. Mg2+ and substrate stabilize the hexameric forms of both variants. The hexameric H136Q- and H140Q-PPases have the same binding affinities for magnesium ion as wild-type, but their hydrolytic activities under optimal conditions are, respectively, 225 and 110% of wild-type PPase, and their synthetic activities, 340 and 140%. The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the catalytic steps in both directions. Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constants for PPi hydrolysis between pH 6 and 9 but drastically decreases their affinities for Mg2PPi and Mg2+. These results prove that His-136 and His-140 are key residues in the dimer interface and show that hexamer formation improves the substrate binding characteristics of the active site.

Amino Acid Sequence↗

PubMed1995

New crystal forms of Escherichia coli and Saccharomyces cerevisiae soluble inorganic pyrophosphatases.

We have obtained new crystal forms of Escherichia coli and Saccharomyces cerevisiae soluble inorganic pyrophosphatase with and without substrate, competitive inhibitor and divalent cation. They diffract to higher resolution than any forms previously reported. The best E. coli crystals are in space group R32 with cell dimensions of 111.4 x 111.4 x 76.6 A and diffract to 2.0 A. The best S. cerevisiae crystals were grown from a mixture of PEG 1000 and 4000 in the presence of metal ions. They are in space group P2(1)2(1)2(1), have cell dimensions of 54.2 x 68.5 x 161.7 A and diffract to 1.8 A.

Journal Article↗

PubMed1995

Cold lability of the mutant forms of Escherichia coli inorganic pyrophosphatase.

The variants of Escherichia coli pyrophosphatase carrying the substitutions Glu20-->Asp, His136-->Gln or His140-->Gln are inactivated, in contrast to the wild-type enzyme, at temperatures below 25 degrees C: their activity measured at 25 degrees C decreases with decreasing the temperature of the stock enzyme solution. The inactivation is completely reversible and is explained by cold-induced dissociation of these hexameric enzymes into less active trimers.

Aspartic Acid↗

PubMed1995

Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis?

Using site-directed mutagenesis, we have completed replacing all 17 putative active site residues of Escherichia coli inorganic pyrophosphatase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydrophobicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for potentially essential residues showed that the effect of the mutation was always to increase the pKa of a basic group essential for both substrate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also had low kcat/Km values. The principal effect seen in the other variant proteins was higher and sharper pH optima; their pH-independent kcat and kcat/Km values changed at most by a factor of 8. Our results suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabilized by coordination to the essential Mg2+ ions. Analyzing our results using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of residues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critical for the activity, possibly by stabilizing the essential hydroxide. Others, including Lys29, Arg43, and Lys142, are more spread out and more positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. coli PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.

Bacterial Proteins↗

PubMed1995

Effect of D97E substitution on the kinetic and thermodynamic properties of Escherichia coli inorganic pyrophosphatase.

Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue forming part of the active site [Cooperman et al. (1992) Trends Biochem. Sci. 17, 262-266]. Here we determine the effect of D97E substitution on several kinetic and thermodynamic properties of E-PPase, including rate and equilibrium constants for enzyme-catalyzed PPi.Pi equilibration at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of substrate, and the Mg2+ and pH dependence of kcat and Km. We find the major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resynthesis on the enzyme, (b) selectively destabilize both the EMg4PPi complex and the transition state between this complex and the EMg2(MgPi)2 complex, (c) raise the pKa of the basic group "essential" for PPi hydrolysis and for productive PPi binding by 1.5 and > 2.2 log units, respectively, (d) distort a site to which Mg2+ binds in the absence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of the two Mg2+ ions that binds to enzyme in the presence of substrate. That this multiplicity of effects arises from a single Asp to Glu substitution suggests, in the absence of any evidence for a generalized structural change, a tightly integrated active site in which the perturbation induced by conservative substitution at a single location can have widespread functional effects.

Bacterial Proteins↗