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T Salminen

Publications and source records attributed to T Salminen.

33 records · Page 2Linked to original sources
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

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

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↗
PubMed1994

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution.

The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9%. The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini. Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology. The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues. Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of pyrophosphatases. Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions. The others, more spreadout and more positively charged, presumably bind substrate. Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently. Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic pyrophosphatases.

Amino Acid Sequence↗
PubMed1992

A survey of FIV antibodies and FeLV antigens in free-roaming cats in the capital area of Finland.

Feline immunodeficiency virus (FIV) was first isolated and identified in 1986. Since then it has been shown to have a worldwide distribution, and the infection generally appears to have reached a state of endemicity. This is the 1st study of FIV-prevalence in Finland. Serum samples of 196 free-roaming cats were tested for antibodies to FIV and FeLV antigens (Feline leukemia virus). With a combined enzyme-linked immunosorbent assay (ELISA), 13 of the cats (6.6%) turned out to be positive for FIV and 2 for FeLV (1.0%). Adult male cats in the capital area of Finland had a FIV prevalence of 24%, a relative proportion 4.7 times higher than that for females.

Animals↗
PubMed1991

Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyr141 are important for the structural integrity.

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases. Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.

Amino Acid Sequence↗
PubMed1990

A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase. Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity.

Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E. coli PPase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B. S. (1990) Biochim. Biophys. Acta 1038, 338-345]. We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104. PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively. This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E. coli PPase. PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase. Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces. Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E. coli PPase.

Amino Acid Sequence↗
PubMed1987

Localization of enkephalin- and neurotensin-like immunoreactivities in cat adrenal medulla.

Enkephalin (ENK)- and neurotensin (NT)-immunoreactivities (IR) were localized in cat adrenal medulla using immunocytochemistry. ENK was localized mainly in adrenaline cells, 70%-80% of which were heavily labelled. About 5%-10% of the noradrenaline cells were ENK-immunoreactive (IR). A dense network of ENK-IR nerves was localized among adrenaline cells. NT was localized only in the noradrenaline cells, 60%-70% of which were immunoreactive. NT- and ENK-IR were localized in separate noradrenaline cells. A dense network of NT-IR nerves was restricted among noradrenaline cells. The present findings confirm that ENK- and NT-like peptides are localized in separate adrenal medullary cells and demonstrate that also the preganglionic nerves are divided in subpopulations according to their neuropeptide content. These nerves have a selective localization among adrenaline and noradrenaline cells and may have physiological significance in the control of secretion of catecholamines and neuropeptides from adrenal medulla.

Adrenal Medulla↗
PubMed1985

Immunohistochemical localization of neurotensin in hamster adrenal medulla.

Neurotensin-like immunoreactivity was localized in nerve fibers and terminals of hamster adrenal medulla at light and electron microscopy using the peroxidase-antiperoxidase method. Numerous varicose neurotensin-immunoreactive nerves and terminals were found among nonlabeled cell groups situated peripherally in the adrenal medulla. Combined formaldehyde-glutaraldehyde (Faglu) fluorescence and immunohistochemistry of the same vibratome section showed that only norepinephrine cells were innervated by neurotensin-immunoreactive nerves. All norepinephrine cells seemed to be innervated by neurotensin-immunoreactive nerves. Neurotensin-immunoreactive nerves disappeared after extrinsic denervation of the adrenal gland. By electron microscopy numerous neurotensin-immunoreactive terminals were seen to make synaptic contacts with norepinephrine cells and with autonomic ganglion cells present in small numbers among norepinephrine cells. In the terminals neurotensin-like immunoreactivity was localized mainly in large dense-cored vesicles, but some precipitates were also associated with small vesicles, diffusely scattered in the axoplasm. The present findings suggest that in the hamster adrenal medulla part of the nerve terminals arising from splanchnic nerves contain neurotensin-like peptide. The functional significance of these nerves in the hamster adrenal medulla remains to be elucidated.

Adrenal Medulla↗
PubMed1985

Localization of enkephalins in adrenaline cells and the nerves innervating adrenaline cells in rat adrenal medulla.

The coexistence of met5- and leu5-enkephalin-like immunoreactivities with catecholamines in the rat adrenal medulla was studied with combined fluorescence microscopy and immunocytochemistry. Both met5- and leu5-enkephalin-like immunoreactivities were localized in few heavily stained adrenaline cells and in a population of nerves innervating adrenaline cells and as well as ganglion cells among the adrenaline cells. Only occasionally single noradrenaline cells exhibited light immunostaining for both enkephalins but no positive fibers could be found around the noradrenaline cells. In electron microscope the immunoreaction was seen in the granules of the adrenaline cells and in the large synaptic vesicles of the nerve terminals around the adrenaline cells. The present findings suggest that enkephalin-like immunoreactivity coexists mainly with adrenaline in rat adrenal medulla and that the enkephalin immunoreactive terminals regulate secretion of adrenaline from rat adrenal medulla.

Adrenal Medulla↗