Inferences about the catalytic domain of P-type ATPases from the tertiary structures of enzymes that catalyze the same elementary reaction.

The machinery to catalyze elementary reactions is conserved, and the number of solved enzyme structures is increasing exponentially. Therefore, structures of enzymes that catalyze phosphate transfer are reviewed, and a supersecondary structure connecting the Walker A sequence to another sequence containing functional amino acids is proposed as an additional signature for the active site. The new signature is used to infer the identity of the P-loop in P-type biological pumps and may be useful in predicting targets for site-directed mutagenesis in other enzymes of unknown structure like the AAA family and ABC transporters.

Oxygen exchange reactions catalyzed by vacuolar H(+)-translocating pyrophosphatase. Evidence for reversible formation of enzyme-bound pyrophosphate.

Vacuolar membrane-derived vesicles isolated from Vigna radiata catalyze oxygen exchange between medium phosphate and water. On the basis of the inhibitor sensitivity and cation requirements of the exchange activity, it is almost exclusively attributable to the vacuolar H(+)-pyrophosphatase (V-PPase). The invariance of the partition coefficient and the results of kinetic modeling indicate that exchange proceeds via a single reaction pathway and results from the reversal of enzyme-bound pyrophosphate synthesis. Comparison of the exchange reactions catalyzed by V-PPase and soluble PPases suggests that the two classes of enzyme mediate P(i)-HOH exchange by the same mechanism and that the intrinsic reversibility of the V-PPase is no greater than that of soluble PPases.

Feasibility of analysing [13C]urea breath tests for Helicobacter pylori by gas chromatography-mass spectrometry in the selected ion monitoring mode.

BACKGROUND: The [13C]urea breath test for Helicobacter pylori is nonradioactive, as well as noninvasive, but few clinical laboratories have the expensive isotope ratio mass spectrometer used for analysis. METHODS: To demonstrate the feasibility of analysing [13C]urea breath tests with a gas chromatograph-mass spectrometer routinely used for drug testing, 13CO2 standards for breath tests and breath samples from patients in a multiple-blind study were analysed. The breath samples were also analysed by isotope ratio mass spectrometry, and the diagnoses were compared with biopsy results. RESULTS: The precision of the enrichment measurements by gas chromatography-mass spectrometry was 1.1 parts per thousand, and the calculated differences in enrichment between standard gases equaled the certified values. The sensitivity (94%), specificity (94%), and percentage agreement (94%) for diagnosis of Helicobacter pylori (n = 34) were as high or higher than for analysis of replicate breath samples by isotope ratio mass spectrometry and comparable to the values reported for diagnosis of the bacterium by other currently accepted tests. CONCLUSIONS: The study demonstrates that a gas chromatograph-mass spectrometer can be used to analyse [13C]urea breath tests, thus potentially lowering the cost of the test and increasing the number of laboratories that can perform the test.

[Inorganic pyrophosphatase--a new enzyme label for biochemical analysis]. / Neorganicheskaia pirofosfataza--novaia fermentnaia metka dlia biokhimicheskogo analiza.

Inorganic pyrophosphatase isolated from Escherichia coli has been proposed as a label in heterogeneous enzyme immunoassays. The enzyme is remarkably stable and insensitive to sodium azide. Enzyme-antibody conjugates were prepared with glutaraldehyde and purified by gel filtration. Enzyme activity was measured by means of a sensitive colour reaction between phosphomolybdate and malachite green. A 5-10-fold increase is sensitivity in terms of absorbance readings was observed compared to peroxidase-based assays. The colour change (yellow/greenish blue) inherent in the use of pyrophosphatase as the labelling agent is highly suitable for visual analysis.

[Use of inorganic pyrophosphatase as a marker in enzyme immunoassays]. / Ispol'zovanie neorganicheskoi pirofosfatazy v kachestve markera v immunofermentnom analize.

A technique of heterogeneous enzyme immunoassay with the E. coli inorganic pyrophosphatase as marker enzyme and Malachite green dye and acidic molybdate as color reagent is developed. Color change (light-yellow/greenish blue) is extremely suitable for visual perception, in some cases making unnecessary the measuring device. Assays with pyrophosphatase are 5-10 times more sensitive than with peroxidase. Further advantages of pyrophosphatase include high thermostability, insensitivity to sodium azide, low value of Michaelis constant (5 microM), substrate stability. Examples are given of use of the pyrophosphatase for assays of human alpha-fetoprotein and immunoglobulin.

Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme.

Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F1,F0-ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F1,F0-ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and Pi as medium ATP concentration was lowered was determined by 18O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the Pi formed as ATP concentration is decreased to the micromolar range. The F1,F0-ATPase from neurospora mitochondria showed an even more pronounced modulation, similar to that of other F1-type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F1,F0-ATPases.

Two pathways for phosphate/water oxygen exchange by yeast inorganic pyrophosphatase.

A scheme of Mg2+ and Pi binding to yeast inorganic pyrophosphatase has been deduced from the concentration dependencies of the rate of oxygen exchange between Pi and water. The exchange reaction requires the binding of MgPi and free Pi (pathway I) or two MgPi (pathway II) in addition to two Mg2+ ions bound in the absence of Pi. Pathway II predominates above 0.16 mM Mg2+. The rate of formation of bound PPi from bound Pi for pathway II is three times as high as that for pathway I. The results suggest that the binding of the fourth Mg2+ ion to pyrophosphatase stimulates its synthetic vs its hydrolytic capability.

Inorganic pyrophosphatase--II. Purification and studies of some properties of the enzyme isolated from thermophylic bacterium Thermus flavus 70 K.

Inorganic pyrophosphatase was isolated from T. flavus in a homogeneous form with a specific activity of 400 U/mg. The enzyme has an isoelectric point 5.0 and consists of 4 subunits each of 24,000 mol. wt. Pyrophosphatase possesses high thermal stability. The enzyme can hydrolyze PPi, ATP and p-nitrophenylphosphate. Kinetic constants of the enzyme's interaction with the metal-activator and metal-substrate complex have been estimated.

Relationships of inosine triphosphate and bicarbonate effects on F1 ATPase to the binding change mechanism.

Two interesting previously reported properties of mitochondrial F1 ATPase have been confirmed and have been examined by 18O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO3- to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO3- was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 microM. The HCO3- activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each Pi formed when 5 or 10 microM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO3- acceleration of ADP departure after a binding change that stops 18O exchange and loosens ADP binding. When ITP concentration was lowered during net ITP hydrolysis by F1 ATPase an increase in water oxygen incorporation into Pi formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [gamma-18O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each Pi formed from the ATP. These results add to evidence for the binding change mechanism for F1 ATPase with sequential participation of catalytic sites.

F1 ATPase from the thermophilic bacterium PS3 (TF1) shows ATP modulation of oxygen exchange.

The ATPase from the ATP synthase of the thermophilic bacterium PS3 (TF1), unlike F1 ATPase from other sources, does not retain bound ATP, ADP, and Pi at a catalytic site under conditions for single-site catalysis [Yohda, M., & Yoshida, M. (1987) J. Biochem. 102, 875-883]. This raised a question as to whether catalysis by TF1 involved alternating participation of catalytic sites. The possibility remained, however, that there might be transient but catalytically significant retention of bound reactants at catalytic sites when the medium ATP concentration was relatively low. To test for this, the extent of water oxygen incorporation into Pi formed by ATP hydrolysis was measured at various ATP concentrations. During ATP hydrolysis at both 45 and 60 degrees C, the extent of water oxygen incorporation into the Pi formed increased markedly as the ATP concentration was lowered to the micromolar range, with greater modulation observed at 60 degrees C. Most of the product Pi formed arose by a single catalytic pathway, but measurable amounts of Pi were formed by a pathway with high oxygen exchange. This may result from the presence of some poorly active enzyme. The results are consistent with sequential participation of three catalytic sites on the TF1 as predicted by the binding change mechanism.

Study of the mechanism of MF1 ATPase inhibition by fluorosulfonylbenzoyl inosine, quinacrine mustard, and efrapeptin using intermediate 18O exchange as a probe.

The mitochondrial F1-ATPase (MF1) is known to be largely or totally inhibited by combination or reaction with one fluorosulfonylbenzoyl inosine (FSBI), quinacrine mustard, or efrapeptin per enzyme. Measurements were made with 18O in attempt to ascertain if the weak catalytic activity remaining after exposure to excess of these reagents was due to retention of some activity by the enzyme modified by these inhibitors. Any such activity could have different characteristics that might be revealed by the distribution of [18O]Pi isotopomers formed from [gamma-18O]ATP. The MF1 inhibited by FSBI showed progressive appearance of two new catalytic pathways as inhibition proceeded. Both pathways appeared to be operative in the enzyme after one beta subunit per enzyme had been modified by FSBI. A high exchange pathway showed no detectable change as ATP concentration was lowered. The lower exchange pathway showed an increase in the amount of exchange with lowering of the ATP concentration, similar to the cooperative behavior observed with the unmodified enzyme. With excess ATP more product was formed by the low exchange pathway, showing that compulsory alternation between two catalytic sites was not retained. The behavior can be explained by the ability of the modified beta subunit to undergo binding changes similar to those occurring in catalysis, with the other two beta subunits catalyzing sluggish hydrolysis by different pathways because of the asymmetry introduced by the modification. Inhibition by quinacrine mustard also resulted in the appearance of two new pathways, somewhat similar to those from FSBI inhibition. In contrast, activity remaining with excess efrapeptin present showed only one pathway like that of the native enzyme. This can be attributed to a low equilibrium concentration of free enzyme and total inhibition of MF1 combined with efrapeptin.

Inorganic pyrophosphatase as a label in heterogeneous enzyme immunoassay.

Inorganic pyrophosphatase from Escherichia coli has been employed as a label in heterogeneous enzyme immunoassays. Enzyme-antibody conjugates were prepared with the use of glutaraldehyde and purified by gel permeation chromatography. Enzyme activity was measured by means of a sensitive one-step color reaction between phosphate, molybdate, and malachite green. The sensitivity in terms of absorbance readings was four to eight times higher than that of peroxidase-based assays. The color change (yellow to greenish blue) inherent in the use of pyrophosphatase as the labeling agent is highly suitable for visual analysis. Other merits of pyrophosphatase include the remarkable stability of the enzyme and its substrate, its compatibility with bacteriostatic agents, and its low Michaelis constant. Examples of the use of phosphatase in the assay of human alpha-fetoprotein and immunoglobulin G are presented.

[Catalytic properties of three isoenzymes possessing pyrophosphatase activity, isolated from baker's yeast]. / Kataliticheskie svoistva trekh izofermentov, obladaiushchikh pirofosfataznoi aktivnost'iu iz pivnykh drozhzhei.

A kinetic study of inorganic pyrophosphatase isolated from brewer's yeast was done. It was shown that all three isoenzymes have the same pH-optimum and specificity with respect to substrate and metal activator. Statistical treatment of the kinetic data yielded equilibrium and catalytical constants, describing enzyme interaction with the metal activator and substrate. The catalytic properties of all three isoenzymes are similar to those of the baker's yeast pyrophosphatase. The fluoride inhibition pattern for inorganic pyrophosphatase from brewer's yeast is similar to that for the baker's yeast enzyme.

[Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase]. / Sravnenie vliianiia ftorida na tri fermenta, gidrolizuiushchie pirofosfat - kisluiu i shchelochnuiu fosfatazy i neorganicheskuiu pirofosfatazu.

The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.

Allosteric regulation of yeast inorganic pyrophosphatase by substrate.

Kinetic and binding studies of yeast inorganic pyrophosphatase (EC 3.6.1.1) revealed a regulatory PPi-binding site. Rate vs substrate concentration dependencies were markedly nonhyperbolic in the range of 0.1-150 microM MgPPi at fixed Mg2+ levels of 0.05-10 mM provided that the enzyme had been preequilibrated with Mg2+. Imidodiphosphate, hydroxymethylenebisphosphonate, and phosphate eliminated the deviations from the Michaelis-Menten kinetics and inhibited PPi hydrolysis in a manner consistent with their binding at both active and regulatory sites. The results agreed with a model in which binding of uncomplexed PPi at the regulatory site markedly increases enzyme affinity for the activating Mg2+ ion. Ultrafiltration studies revealed the binding of at least 3 mol of the inhibitory hydroxymethylenebisphosphonate and of 2 mol of noninhibitory methylenebisphosphonate per mole of the dimeric enzyme.

A proposal for the Mg2+ binding site of P-type ion motive ATPases and the mechanism of phosphoryl group transfer.

Mutations of D586 in the DPPR sequence of sodium pump decrease the enzyme's affinity for inorganic phosphate [Farley R. A., Heart, E., Kabalin, M., Putnam, D., Wang, K., Kasho, V. N., and Faller, L. D. (1997) Biochemistry 36, 941-951]. Therefore, it was proposed that D586 coordinates the Mg2+ required for catalytic activity. This hypothesis is tested (1) by determining the substrate for catalysis of 18O exchange between inorganic phosphate and water and (2) by comparing conserved amino acid sequences in P-type pumps with the primary structures of enzymes of known tertiary structure that catalyze phosphoryl group transfer. From the isotope exchange data, it is concluded that the Mg2+-dependent and Na+- and K+-stimulated ATPase binds Mg2+ before inorganic phosphate. Sequence homology is demonstrated between the conserved DPPR and MV(I,L)TGD sequences of P-type pumps and two conserved adenylate kinase sequences that coordinate Mg2+ and/or bind nucleotide in the crystal structure of the yeast enzyme. A model for the Mg2+ site of P-type pumps and the mechanism of phosphoryl group transfer is proposed and tested by demonstrating that the conserved sequences are also structurally homologous.

[Isolation and catalytic properties of the soluble monomeric form of inorganic pyrophosphatase from baker's yeast]. / Poluchenie i kataliticheskie svoistva rastvorimoi monomernoi formy neorganicheskoi pirofosfatazy iz pekarskikh drozhzhei.

Data from sedimentation analysis suggest that modification of about 40% of free amino groups of inorganic pyrophosphatase by maleic anhydride, pH 10.5, results in a loss of the enzyme ability to form dimers at neutral values of pH. The specific activity of monomeric pyrophosphatase is 50-80% of that of the dimeric form. The monomer has a pH optimum of about 7, requires metal ions for activation of both enzyme and substrate and is capable of exergonic synthesis of PPi in the active center. The enzyme binding to PPi is strongly stabilized by fluoride. The experimental data indicate that the individual subunit of inorganic pyrophosphatase possesses all the main catalytic properties of native dimeric molecule.

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.

Rates of elementary steps catalyzed by rat liver cytosolic and mitochondrial inorganic pyrophosphatases in both directions.

We have investigated kinetics of pyrophosphate synthesis and phosphate-water oxygen exchange catalyzed by rat liver cytosolic and mitochondrial pyrophosphatases in the presence of Mg2+ as cofactor. A common kinetic model derived for these reactions implies that they involve formation of enzyme-bound pyrophosphate and proceed through two parallel pathways: pathway I, utilizing two magnesium phosphate molecules, and pathway II, utilizing both magnesium phosphate and free phosphate. Pyrophosphate formation is greatly facilitated in the active sites of both pyrophosphatases ([E.PPi]/[E.2Pi] = 0.11-0.24) compared to solution. The rate constants for PPi binding/release, bound PPi hydrolysis/synthesis, and two Pi binding/release steps catalyzed by cytosolic and mitochondrial pyrophosphatases were enumerated for pathway I. There is no unique rate-limiting step for pathway I for both enzymes in either direction. A modulating effect of magnesium phosphate on the oxygen exchange is observed with the cytosolic pyrophosphatase, explicable in terms of an allosteric phosphate-binding site or random-order release of two phosphate molecules from the active site. A remarkable feature of these mammalian pyrophosphatases versus their microbial counterparts is their high efficiency in pyrophosphate synthesis. The turnover numbers in the direction of synthesis are 14 and 9.3 s-1 for the cytosolic and mitochondrial enzymes, respectively (9 and 16% relative to hydrolysis turnover numbers). The results demonstrate that the enzyme-catalyzed synthesis of pyrophosphate, the simplest high-energy polyphosphate, can proceed at a high rate in the absence of an external energy input, such as that provided by protonmotive force in membrane systems.