Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.     

Functional interrelationships in the alkaline phosphatase superfamily: phosphodiesterase activity of Escherichia coli alkaline phosphatase

Escherichia coli alkaline phosphatase (AP) is a proficient phosphomonoesterase with two Zn(2+) ions in its active site. Sequence homology suggests a distant evolutionary relationship between AP and alkaline phosphodiesterase/nucleotide pyrophosphatase, with conservation of the catalytic metal ions. Furthermore, many other phosphodiesterases, although not evolutionarily related, have a similar active site configuration of divalent metal ions in their active sites. These observations led us to test whether AP could also catalyze the hydrolysis of phosphate diesters. The results described herein demonstrate that AP does have phosphodiesterase activity: the phosphatase and phosphodiesterase activities copurify over several steps; inorganic phosphate, a strong competitive inhibitor of AP, inhibits the phosphodiesterase and phosphatase activities with the same inhibition constant; a point mutation that weakens phosphate binding to AP correspondingly weakens phosphate inhibition of the phosphodiesterase activity; and mutation of active site residues substantially reduces both the mono- and diesterase activities. AP accelerates the rate of phosphate diester hydrolysis by 10(11)-fold relative to the rate of the uncatalyzed reaction [(k(cat)/K(m))/k(w)]. Although this rate enhancement is substantial, it is at least 10(6)-fold less than the rate enhancement for AP-catalyzed phosphate monoester hydrolysis. Mutational analysis suggests that common active site features contribute to hydrolysis of both phosphate monoesters and phosphate diesters. However, mutation of the active site arginine to serine, R166S, decreases the monoesterase activity but not the diesterase activity, suggesting that the interaction of this arginine with the nonbridging oxygen(s) of the phosphate monoester substrate provides a substantial amount of the preferential hydrolysis of phosphate monoesters. The observation of phosphodiesterase activity extends the previous observation that AP has a low level of sulfatase activity, further establishing the functional interrelationships among the sulfatases, phosphatases, and phosphodiesterases within the evolutionarily related AP superfamily. The catalytic promiscuity of AP could have facilitated divergent evolution via gene duplication by providing a selective advantage upon which natural selection could have acted.     

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.     

A microfluorometric method for alkaline phosphatase: Application to the various segments of the nephron

A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l-bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.     

Lack of relationship between activity of intestinal alkaline phosphatase and calcium or phosphate absorption

The effects of vitamin D3 and the aqueous extract of Solanum malacoxylon on intestinal alkaline phosphatase and tissue phosphate content were studied on rachitic chicks treated with large doses of ethane-1-hydroxy-1,1 diphosphonate (EHDP). The EHDP treatment blocks the increase of intestinal calcium or phosphate absorption induced by the vitamin D3, while it has no effects on the rise of intestinal alkaline phosphatase activity or the increment in tissue phosphate content. The lack of correlation between the increment of alkaline phosphatase and that of Ca or phosphate absorption in vitamin D3 plus EHDP treated chicks excludes a participation of the alkaline phosphatase in the mechanism of Ca or P intestinal absorption. The Ca or phosphorus absorption are elicited specifically by 1,25-(OH)2-D3, while alkaline phosphatase activity and phosphate tissue concentration respond to a broader spectrum of stimuli.     

Low activity of alkaline phosphatase in two eutrophic reservoirs

We studied recycling of phosphate by enzymatic hydrolysis in two temperate very eutrophic reservoirs. To assess the potential importance of phosphate regeneration by alkaline phosphatase, we determined the activity of this enzyme in lake water concomitantly with the determinations of the concentrations of phosphomonoesters, soluble reactive phosphate, total soluble phosphate and total phosphate. Contrary to our expectations for such productive waters where algal blooms are frequent, during the study period this process of phosphate regeneration was not significant, probably because the product of hydrolysis (contained in the soluble reactive phosphate fraction) was always abundant. We conclude that, in spite of what has been observed repeatedly in natural lakes with similar trophic characteristics, the readily available fraction of phosphate in these reservoirs is large and for that reason alkaline phosphatase production is low. Therefore hydrolysis by this enzyme is not significant for growth. What seems intriguing is the small amount of phosphomonoesters found in the water; with no phosphatase activity this phosphate fraction should always be high, unless hydrolysis takes place either during phosphomonoester release or later due to their instability.     

Studies on the mechanism of action of the alkaline phosphatase from Dictyostelium discoideum

The Dictyostelium discoideum alkaline phosphatase was investigated kinetically in an attempt to elucidate its mechanism of action. Analysis of the hydrolysis of p-nitrophenyl phosphate by stopped-flow spectrophotometry revealed biphasic kinetics, suggesting a double displacement enzyme mechanism. Furthermore, Tris stimulated activity in an uncompetitive manner, a result that was consistent with this interpretation. The enzyme was inhibited reversibly by phosphate at low ionic strength, but the inhibition was irreversible at high ionic strength and the latter effect was enhanced at alkaline pH values. These results indicate that high ionic strength and alkaline pH conditions bring about a conformational change that renders the enzyme susceptible to irreversible inhibition by phosphate.     

A Limulus glucose-6-phosphatase with phosphotransferase activity characteristic of vertebrate liver microsomes. Its possible evolutionary significance.

1. Limulus hepatopancreas, coxal glands and intestine contain a particulate enzyme which can synthesize glucose 6-phosphate from glucose and inorganic pyrophosphate or carbamyl phosphate as well as hydrolyze glucose 6-phosphate. This has been clearly differentiated from hydrolysis by lysosomal or soluble phosphatases. 2. The enzyme resembles vertebrate glucose-6-phosphatase in its specific anatomical distribution, pH optimum, kinetic properties, donor specificity and phospholipid dependence, as indicated by its satency and lability to detergent treatment. 3. A variety of other invertebrates tested exhibited little or no PPi-glucose phosphotransferase activity with these properties. A similar phosphotransferase activity of lobster hepatopancreas had somewhat different kinetic properties and pH optimum. 4. The hypothesis that a specific glucose-6-phosphatase is to be found only in those animals which utilize free glucose as an important circulating form of energy is presented and discussed. It appears that a variety of transport compounds, such as trehalose and glucose, was tried at the evolutionary level of the Arthropods.     

Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.     

Dipalmitoyl-phosphatidylcholine/phospholipase D interactions investigated with polarization-modulated infrared reflection absorption spectroscopy

The hydrolysis of 1,2-dipalmitoylphosphatidylcholine (DPPC) catalyzed by Streptomyces chromofuscus phospholipase D (PLD) has been investigated using monolayer techniques and polarization-modulated infrared absorption reflection spectroscopy. The spectroscopic analysis of the phosphate groups provides a quantitative estimation of the hydrolysis yield. The hydrolysis kinetics was investigated in dependence on the phase state of the lipid monolayer. It was found that PLD exhibits maximum activity in the liquid-expanded phase, whereas PLA2 has its activity maximum in the two-phase region. A lag phase was observed in all experiments indicating that small amounts of the hydrolysis product 1,2-dipalmitoylphosphatidic acid (DPPA) are needed for initiating the fast hydrolysis reaction. Higher concentrations of DPPA inhibit the hydrolysis. The critical inhibition concentration of DPPA is a function of the monolayer pressure.     

Biosynthesis of dolichyl phosphate: characterization and site of synthesis in algae

This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system.

A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate.

The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [¹⁴C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C₉₀ to C₁₀₅, C₉₅ and C₁₀₀ being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed.

The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate.

     

Effect of teichoic acid on resistance to the membrane-lytic agent of Streptococcus zymogenes

Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Effect of teichoic acid on resistance to the membrane-lytic agent of Streptococcus zymogenes. J. Bacteriol. 92:1623-1631. 1966.-The resistance of Streptococcus zymogenes to its own lytic agent has been shown to be due to the production of a specific, inhibitory teichoic acid. A survey of streptococcal strains showed that only strains resistant to the lytic agent produced the specific inhibitor. In addition, the inhibitor can be removed from spheroplasts of resistant strains, thereby making them sensitive to the lysin. Throughout the early part of the growth cycle, the inhibitor is associated with the cell and cannot be found in the medium. During late logarithmic phase, however, the inhibitor is released into the medium by the cells, and therefore is a contributing factor to the apparent lability of the lytic agent. The purified, inhibitory teichoic acid contains ribitol, phosphate, glucose, and d-alanine. The alkaline lability of the biological activity of the teichoic acid was correlated with the hydrolysis of the d-alanine. A streptococcal strain which is sensitive to the membrane-lytic agent produced an inactive ribitol teichoic acid which lacks the ester-linked d-alanine, whereas a lysin-resistant mutant of this strain produces a teichoic acid which contains d-alanine and which has inhibitory activity.     

Relationship between alkaline phosphatase and neomycin formation in Streptomyces fradiae

Studies on phosphatase activity of Streptomyces fradiae 3535 grown in three different media indicate that neomycin formation varies directly with enzyme activity, sodium nitrate-maltose-mineral salts medium giving the highest yields of alkaline phosphatase and neomycin. S. fradiae contains more than one alkaline phosphatase and the phosphatase responsible for hydrolysis of neomycin phosphate appears to be substrate specific. The same enzyme apparently hydrolyses both the N-P and P-O-P bonds of neomycin pyrophosphate. The enzyme is stimulated by Ca(2+), is inactive at a pH below 7 and is inhibited by EDTA. Enzymic activity increases when mycelia are incubated in mineral salts medium, but decreases when phosphate or glucose is included in the medium, although the latter is more effective. The inhibitory effect of EDTA on neomycin formation by resting mycelia is completely reversed by Ca(2+).     

Formation of the Digestive-Transport Conveyer of Carbohydrates in Rat Ontogenesis

The absorption of free glucose and glucose released from hydrolysis of different oligosaccharides has been studied using the procedure of perfusion of the isolated segment of the small intestine of rat pups, with the simultaneous determination of activities of intestinal disaccharidases by glucoseoxidase method. It has been established that: (1) the active transport of glucose in the small intestine is sufficiently pronounced during the breast feeding and gradually decreases by the time of transition of the pups to the definitive nutrition; (2) at this period, activities of lactase and of lactase-transport complexes are rather high, with the low level of activities of maltase and of maltase-transport ensembles, or complexes; (3) during the transition to the definitive feeding, simultaneously with the lactase activity repression, the activity of the lactase-transport ensembles is reduced, and digestive-transport complexes are formed, which provide high rates of hydrolysis of carbohydrates, the main component of the food.     

Glucose-specific permease of the bacterial phosphotransferase system: phosphorylation and oligomeric structure of the glucose-specific IIGlc-IIIGlc complex of Salmonella typhimurium

The glucose-specific membrane permease (IIGlc) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates active transport and concomitant phosphorylation of glucose. The purified permease has been phosphorylated in vitro and has been isolated (P-IIGlc). A phosphate to protein stoichiometry of between 0.6 and 0.8 has been measured. Phosphoryl transfer from P-IIGlc to glucose has been demonstrated. This process is, however, slow and accompanied by hydrolysis of the phosphoprotein unless IIIGlc, the cytoplasmic phosphoryl carrier protein specific to the glucose permease (IIGlc) of the PTS, is added. Addition of unphosphorylated IIIGlc resulted in rapid formation of glucose 6-phosphate with almost no hydrolysis of P-IIGlc accompanying the process. A complex of IIGlc and IIIGlc could be precipitated from bacterial cell lysates with monoclonal anti-IIGlc immunoglobulin. The molar ratio of IIGlc:IIIGlc in the immunoprecipitate was approximately 1:2. Analytical equilibrium centrifugation as well as chemical cross-linking showed that purified IIGlc itself is a dimer (106 kDa), consisting of two identical subunits. These results suggest that the functional glucose-specific permease complex comprises a membrane-spanning homodimer of IIGlc to which four molecules of IIIGlc are bound on the cytoplasmic face.     

Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels

A sensitive staining method for alkali-labile phosphoproteins has been developed. As little as 0.2 nmol bound P/mm2 can be detected. The procedure is based on alkaline hydrolysis, phosphate capture, and formation of an insoluble rhodamine B-phosphomolybdate complex. A further modification for the qualitative detection of alkaline phosphatase activity on polyacrylamide gels is proposed. During incubation, the released Pi is precipitated as lead phosphate and subsequently stained with rhodamine B.     

UDP-Glucose-Dependent Sucrose Translocation in Tonoplast Vesicles from Stalk Tissue of Sugarcane

Tonoplast vesicles isolated from stalk parenchyma tissue of sugarcane plants transport sucrose via a uridine diphosphate glucose (UDPGlc)-dependent group translocator. No sucrose transport via an ATP-dependent system could be detected. The products of UDPGlc uptake in the vesicles were sucrose and sucrose phosphate which, upon hydrolysis with alkaline phosphatase and invertase, showed that both hexose moieties are derived from UDPGlc.     

Synthesis of mannosyl cellobiose diphosphate prenol in Acetobacter xylinum

The enzymatic synthesis of a β-mannosyl (1 → 3) β-glucosyl (1 → 4) α-glucose-1-pyrophosphate-prenol (allylic) by Acetobacter xylinum preparations is described. Glucose pyrophosphate lipid, already known to be formed from UDP-glucose and endogenous phosphate lipid, is demonstrated to accept another glucose from UDP-glucose to give a cellobiose pyrophosphate lipid. The latter in turn accepts mannose from GDP-mannose to form a mannosyl cellobiose pyrophosphate lipid. The structure of the trisaccharide and the way it is linked to the lipid moiety were established by enzymatic and chemical methods such as mild alkaline and acid hydrolysis, phenol treatment, partial acid hydrolysis and acetolysis, periodate oxidation, borohydride reduction, and treatments with glycosidases. The α-unsaturated, polyprenolic nature of the lipid was inferred from and confirmed by the reaction between UDP-glucose and ficaprenol monophosphate to give glucose pyrophosphate ficaprenol, which had the same properties as the glucose pyrophosphate lipid formed from the endogenous acceptor. The allylic structure proposed for the endogenous acceptor is suggested by the lability to phenol treatment and catalytic reduction of its glycosylated derivatives. The enzyme preparation also synthesizes a β-mannose phosphate prenol (allylic), which does not seem to participate in the trisaccharide synthesis. The possible role of these sugar prenols in the synthesis of exopolysaccharides is considered.     

Purification and characterization of a repressible alkaline phosphatase from Thermus aquaticus.

A repressible alkaline phosphatase has been isolated from the extreme bacterial thermophile, Thermus aquaticus. The enzyme can be derepressed more than 1,000-fold by starving the cells for phosphate. In derepressed cells, nearly 6% of the total protein in a cell-free enzyme preparation is alkaline phosphatase. The enzyme was purified to homogeneity as judged by disc acrylamide electrophoresis and sodium dodecyl sulfate electrophoresis. By sucrose gradient centrifugation it was established that the enzyme has an approximate molecular weight of 143,000 and consists of three subunits, each with a molecular weight of 51,000. Tris buffer stimulates the activity of the enzyme, which has a pH optimum of 9.2. The enzyme has a broad temperature range with an optimum of 75-80 degrees. The enzyme catalyzes the hydrolysis of a wide variety of phosphorylated compounds as do many of the mesophilic alkaline phosphatases. The Michaelis constant(Km) for the enzyme is 8.0 X 10(-4) M. Amino acid analysis of the protein revealed little in the amino acid composition to separate it from other mesophilic enzymes which have been previously studied.     

Pharmacologic properties of a phosphate ester of dopamine

The 3 or 4 phosphate ester of dopamine (PD) was hydrolyzed by homogenates of rat tissues to give inorganic phosphate (Pi) and dopamine. The rate of hydrolysis of PD by kidney homogenates was increased by exogenous MgCl2 but not CaCl2 or KCl. The activity of brain, heart or liver homogenates was insensitive to the added salts. Several lines of evidence indicate that alkaline phosphatase activity contributes to the high rate of PD hydrolysis by the kidney but not brain homogenate. The intravenous infusion of PD at 12 mumole/kg in one hr to anesthetized rats increased the dopamine content of the plasma, kidney and heart without altering brain or liver dopamine. The results suggest that PD may be more effective than dopamine for increasing dopamine levels of the kidney. In addition, the hydrolysis of PD by brain homogenates, which is independent of alkaline phosphatase activity, suggests that specific enzymes exist for the metabolism of PD.