Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach

Two major α-glucan phosphorylases (I and II) from leaves of the C₄ plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C₃ plant spinach. The two α-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K_m = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C₃ plant spinach with mesophyll cell phosphorylase of the C₄ plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.     

Studies on phosphorylase isozymes in lower vertebrates. Purification and properties of lamprey phosphorylase.

Skeletal muscle phosphorylase b has been purified from lamprey, Entosphenus japonicus, to a state of homogeneity as judged by the criterion of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme was completely dependent on AMP for activity and converted into the a form by rabbit muscle phosphorylase kinase in the presence of ATP and Mg²⁺. The subunit molecular weight determined by SDS-gel electrophoresis was 94,000 ± 1,600 (SE). The enzyme activity was stimulated by Na₂SO₄, but was not affected by mercaptoethanol. The K_m values of the a form for glucose 1-phosphate and glycogen were 3.5 mm and 0.13%, respectively, and those of the b form for glucose 1-phosphate, glycogen, and AMP were 15 mm, 0.4%, and 0.1 mm, respectively. These values were smaller than those reported with lobster phosphorylase and greater than those reported with mammalian skeletal muscle phosphorylases. Electrophoretic and immunological studies have indicated that lamprey phosphorylase b exists as a single molecular form in skeletal muscle, heart, brain, and kidney. Rabbit antibody against lamprey phosphorylase cross-reacted with phosphorylases from skate and shark livers more intensely than with those from skeletal muscles.     

Characterization of the spinach leaf phosphorylases

The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V_max for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K_m for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K_m values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V_max values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K_m values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K_m value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity.     

Starch phosphorylase: Role in starch metabolism and biotechnological applications

The α-glucan phosphorylases of the glycosyltransferase family are important enzymes of carbohydrate metabolism in prokaryotes and eukaryotes. The plant α-glucan phosphorylase, commonly called starch phosphorylase (EC 2.4.1.1), is largely known for the phosphorolytic degradation of starch. Starch phosphorylase catalyzes the reversible transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of α-1,4-d-glucan chains with the release of phosphate. Two distinct forms of starch phosphorylase, plastidic phosphorylase and cytosolic phosphorylase, have been consistently observed in higher plants. Starch phosphorylase is industrially useful and a preferred enzyme among all glucan phosphorylases for phosphorolytic reactions for the production of glucose-1-phosphate and for the development of engineered varieties of glucans and starch. Despite several investigations, the precise functional mechanisms of its characteristic multiple forms and the structural details are still eluding us. Recent discoveries have shed some light on their physiological substrates, precise biological functions, and regulatory aspects. In this review, we have highlighted important developments in understanding the role of starch phosphorylases and their emerging applications in industry.     

Purification and characterization of an acid phosphatase from Trichoderma harzianum

An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55°C. The enzyme retained 60% of its activity after incubation at 55°C for 60 min. The K _m and V _max values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min^?1, respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.     

Purification and characterization of a novel alkaline β-1,3-1,4-glucanase (lichenase) from thermophilic fungus Malbranchea cinnamomea

A novel alkaline β-1,3-1,4-glucanase (McLic1) from a thermophilic fungus, Malbranchea cinnamomea, was purified and biochemically characterized. McLic1 was purified to homogeneity with a purification fold of 3.1 and a recovery yield of 3.7 %. The purified enzyme was most active at pH 10.0 and 55 °C, and exhibited a wide range of pH stability (pH 4.0–10.0). McLic1 displayed strict substrate specificity for barley β-glucan, oat β-glucan and lichenan, but did not show activity towards other tested polysaccharides and synthetic p-nitrophenyl derivates, suggesting that it is a specific β-1,3-1,4-glucanase. The K _m values for barley β-glucan, oat β-glucan and lichenan were determined to be 0.69, 1.11 and 0.63 mg mL^?1, respectively. Moreover, the enzyme was stable in various non ionic surfactants, oxidizing agents and several commercial detergents. Thus, the alkaline β-1,3-1,4-glucanase may have potential in industrial applications, such as detergent, paper and pulp industries.     

Cause of the abnormal acidity of ionogenic groups of the lysozyme active center in cell wall lysis <Emphasis Type="Italic">Micrococcus lysodeicticus</Emphasis>

The influence of pH within the range 6.9–10.0 on the kinetic parameters of Micrococcus lysodeicticus cell lysis catalyzed by hen egg lysozyme has been studied at 25°C and 37°C. The effective pK _b values have been calculated for the group determining lysozyme catalytic activity. The ΔH _ion value indicates that this group is a carboxyl, although its pK (9.15 at 25°C) is far beyond the range characteristic of carboxylic groups. The cause of this abnormal pK _b value is supposed to be the strong negative charge of the bacterial cell wall. As a result, the enzyme, which catalyzes the hydrolysis of N-acetylglucosamine-N-acetylmuramic acid copolymer, operates in a highly acidic microenvironment.     

Human placental alkaline phosphatase: Effects on conformation by ligands which alter catalytic activity

The conformation of human placental alkaline phosphatase (EC 3.1.3.1) has been studied using the spectroscopic structural probes of pH difference spectroscopy, solvent perturbation difference spectroscopy, and circular dichroism. Of the 37 ± 1 tyrosine residues in placental alkaline phosphatase (PAP), 5 ± 1 residues are observed by pH difference spectroscopy to be “free” and presumed to be located on the surface of the enzyme molecule. The ionization of these 5 “free” tyrosyl groups is not time dependent and is reversible with a pK_app of 10.29. The remaining 32 ± 1 tyrosines are considered “buried” and ionization is observed to be both time dependent and irreversible. Treatment of the enzyme with 4 m guanidine-hydrochloride normalizes all 37 ± 1 tyrosine residues (pK_app = 10.08). The difference pH titration studies thus provide spectrophotometric evidence for a change in molecular conformation of PAP in the pH region of 10.5. Using solvent perturbation difference spectroscopy and circular dichroism, the local environments of tyrosine and tryptophan residues were elucidated for the native enzyme and the enzyme in the presence of ligands that influence catalytic function: inorganic phosphate (competitive inhibitor), l-phenylalanine (uncompetitive inhibitor), d-phenylalanine (noninhibitor). and Mg²⁺ ion (activator). The spectral observations from these studies led to the following interpretations: (i) the binding of inorganic phosphate, a competitive inhibitor, induces a conformational change in the enzyme that may alter the active site and thereby decrease enzyme catalytic function; (ii) perturbation with l-phenylalanine gives spectral results indicating a conformational change consistent with the postulate that this uncompetitive inhibitor prevents the dissociation of the phosphoryl enzyme intermediate; and (iii) Mg²⁺ ion causes a slight separation of the enzyme subunits, which could increase accessibility to the active site and, thus, enzyme activity.     

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase

Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90–100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Human liver acid phosphatases: Purification and properties of a low-molecular-weight isoenzyme

A low-molecular-weight human liver acid phosphatase was purified 2580-fold to homogenity by a procedure involving ammonium sulfate fractionation, acid treatment, and SP-Sephadex ion-exchange chromatography with ion-affinity elution. The purified enzyme contains a single polypeptide chain and has a molecular weight of 14,400 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of this enzyme (E) is reported. A pH dependence study using p-nitrophenyl phosphate as a substrate (S) revealed the effect of substrate ionization (pK_a 5.2) and the participation of a group in the ES complex having a pK_a value of 7.8. The enzyme is readily inactivated by sulfhydryl reagents such as heavy metal ions. Alkylation of the enzyme with iodoacetic acid and iodoacetamide causes complete inactivation of the enzyme and this inactivation is prevented by the presence of phosphate ion. The enzyme is also inactivated by treatment with diethyl pyrocarbonate; protection against this reagent is afforded by phosphate ion. The substrate specificity of this enzyme is unusual for an acid phosphatase. Of the many alkyl and aryl phosphomonoesters tested, the only possibly physiological substrate hydrolyzed by this enzyme was flavin mononucleotide, which exhibits a V which is 3-fold larger at pH 5.0 and 6-fold larger at pH 7.0 than that for p-nitrophenyl phosphate. However, the enzyme also catalyzes the hydrolysis of acetyl phosphate at pH 5.0 with a velocity eight times larger than that reported for an acyl phosphatase from human erythrocytes.     

Purification and molecular characterization of a novel diadenosine 5′,5′′′-P1,P4-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K_m values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.     

Protonation of kanamycin A: Detailing of thermodynamics and protonation sites assignment

Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of ¹H-¹³C-HSQC spectra at variable pH in D₂O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pK_a values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pK_a 7.98, in ring B by ionization of two amino groups with pK_a 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pK_a 9.19 and some atoms felt ionization of a second group with pK_a 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pK_a 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

Enzymatic transglycosylation of natural and modified nucleosides by immobilized thermostable nucleoside phosphorylases from Geobacillus stearothermophilus

Natural and modified purine nucleosides have been synthesized using the recombinant thermostable enzymes purine nucleoside phosphorylase II (E. C. 2.4.2.1) and pyrimidine nucleoside phosphorylase (E. C. 2.4.2.2) from Geobacillus stearothermophilus B-2194. The enzymes were produced in recombinant E. coli strains and covalently immobilized on aminopropylsilochrom AP-CPG-170 after heating the cell lysates and the removal of coagulated thermolabile proteins. The resulting preparations of thermostable nucleoside phosphorylases retained a high activity after 20 reuses in nucleoside transglycosylation reactions at 70–75°C with a yield of the target products as high as 96%. Owing to the high catalytic activity, thermal stability, the ease of application, and the possibility of repeated use, the immobilized preparations of thermostable nucleoside phosphorylases are suitable for the production of pharmacologically important natural and modified nucleosides.     

Development and application of a screening assay for glycoside phosphorylases

Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme’s substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl β-glucosides and phenyl β-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.     

Activation volumes for horseradish peroxidase compound ii reactions

The activation volumes for the reactions of horseradish peroxidase compound II with L-tyrosine. 3-iodo-L-tyrosine. p-aminobenzoic acid and ferrocyanide were determined by using a high-pressure stopped-flow technique at 25°C and pH 7. For the tyrosines, the solvent electrostriction accompanying substrate ionization and H⁺ transfer from the substituted phenol to a basic group of the enzyme can account for the observed negative activation volumes. For p-aminobenzoic acid a simple electron transfer without H⁺ transfer appears to occur. The positive activation volume for ferrocyanide may be explained in terms of electron transfer associated with a large change in electrostriction of the inorganic redox couple.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

On the amino acids involved in the ATPase site of mitochondrial F1 and the implication of the subunit C.

The data on the pH dependence of the K_m for Mg-ATP and the V_m of the ATPase of pig heart mitochondrial F₁ indicate the presence of two groups of different pK's which modify the enzyme activity. The first pK at pH 9.6 ± 0.2 may be related to the possible presence of arginine and/or tyrosine residues in the ATPase site; the second pK at pH 7.2 ± 0.2 could be due to the presence of a histidine residue in the ATPase site or to the involvement of amino groups in the ATPase site. The inhibition induced by photooxidation in the presence of Rose Bengal is not pH dependent in the pH range corresponding to the pK of histidine. The inhibition induced by diethylpyrocarbonate cannot be reversed by hydroxylamine and the characteristics of this inhibition rather correspond to the reaction of the inhibitor with amino groups. Pyridoxal phosphate also inhibits the ATPase activity of F₁ by reaction with amino groups. The presence of ATP or phosphate partially protects against the inhibition induced by diethylpyrocarbonate or pyridoxal phosphate, which indicates that amino groups may be directly or indirectly involved in the binding of nucleotide and phosphate to F₁. Glutaraldehyde also inhibits the enzyme by reacting with amino groups and inducing a crosslinking of the subunits. The disappearance of subunit C is well correlated with the decrease of ATPase activity, indicating that subunit C is essential in the ATPase activity.     

Standard free energy maps for the hydrolysis of ATP as a function of pH and metal ion concentration: comparison of metal ions

The observed equilibrium constant K_obs for the hydrolysis of ATP to ADP and inorganic phosphate has been calculated as a function of pH and metal ion concentration pM (- log [M]) at 25 °C and μ = 0.2 with the use of literature values of the acid dissociation and complex dissociation constants for the phosphates.The resulting standard free energy changes ΔG °′ are presented by means of contour diagrams for the range pH 4–10 and pM 1–7. These maps summarize the results of some 1900 calculations per diagram, and clearly simulate a differential effect of the metal ions of interest, including Mg²⁺, Ca²⁺, Sr²⁺, Mn²⁺, Li⁺, Na⁺ and K⁺, on the equilibrium hydrolysis of ATP.