Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

The influence of inorganic phosphate on the activity of adenosine kinase

The enzyme adenosine kinase (AK; EC 2.7.1.20) shows a dependence upon inorganic phosphate (Pi) for activity. The degree of dependence varies among enzyme sources and the pH at which the activity is measured. At physiological pH, recombinant AK from Chinese hamster ovary (CHO) cells and AK from beef liver (BL) show higher affinities for the substrate adenosine (Ado), larger maximum velocities and lower sensitivities to substrate inhibition in the presence of Pi. At pH 6.2, both BL and CHO AK exhibit almost complete dependence on the presence of Pi for activity. The data show that both enzymes exhibit increasing relief from substrate inhibition upon increasing Pi and the inhibition of BL AK is almost completely alleviated by the addition of 50 mM Pi. The affinity of CHO AK for Ado increases asymptotically from K(m) 6.4 microM to a limit of 0.7 microM upon the addition of increasing Pi from 1 to 50 mM. The concentration of Ado necessary to invoke substrate inhibition also increases asymptotically from K(i) 32 microM to a limit of 69 microM at saturating concentrations of phosphate. In the presence of increasing amounts of Pi, the maximal velocity of activity increases hyperbolically. The effect that phosphate exerts on AK may be either to protect the enzyme from inactivation at high adenosine and H(+) concentrations or to stabilize substrate binding at the active site.     

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.     

Reconstitution of the phosphoglycerate transport protein of Salmonella typhimurium

Operation of the phosphoglycerate transport protein (PgtP) of Salmonella typhimurium has been studied in proteoliposomes by using a technique in which membrane protein is solubilized and reconstituted directly from small volumes of cell cultures. When protein from induced cells was reconstituted into phosphate (Pi)-loaded proteoliposomes, it was possible to demonstrate a PgtP-mediated exchange of internal and external phosphate. For this homologous Pi:Pi antiport, kinetic analysis indicated a Michaelis constant (Kt) of 1 mM and a maximal velocity of 26 nmol/min mg of protein; arsenate inhibited with a Ki of 1.3 mM, suggesting that PgtP did not discriminate between these two inorganic substrates. Pi-loaded proteoliposomes also accumulated 3-phosphoglycerate and phosphoenolpyruvate, establishing for each of them a concentration gradient (in/out) of about 100-fold; phosphoenolpyruvate (Ki = 70 microM) rather than 3-phosphoglycerate (Kt = 700, Ki = 900 microM) was the preferred substrate for these conditions. We also concluded that such heterologous exchange was a neutral event, since its rate and extent were unaffected by the presence of a protonophore and unresponsive to the imposition of a membrane potential (positive or negative inside). In quantitative work, we found a stoichiometry of 1:1 for the exchange of Pi and 3-phosphoglycerate, and given an electroneutral exchange, this finding is most easily understood as the overall exchange of divalent Pi against divalent phosphoglycerate. These experiments establish that PgtP functions as an anion exchange protein and that it shares important mechanistic features with the Pi-linked antiporters, GlpT and UhpT, responsible for transport of glycerol 3-phosphate and hexose 6-phosphates into Escherichia coli.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

The kinetics of a purified form of 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (tryptophan) from Neurospora crassa.

1. A method is described for the purification of a form of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) that probably differs from that of the native enzyme. 2. The kinetics of the reaction catalysed by 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (tryptophan) shows that the reaction proceeds via a ping-pong bi-bi mechanism, with activation by phosphoenolpyruvate (P-Prv), the first substrate, and inhibition by erythrose 4-phosphate (Ery-P) the second substrate. At low substrate concentrations, KP-Prv is 0.1 mM and KEry-P is 0.13 mM. 3. The substrates phosphoenolpyruvate and erythrose 4-phosphate and the product inorganic phosphate can protect the purified enzyme against heat denaturation, whereas the inhibitor, tryptophan, has no effect, although it binds to the enzyme in the absence of other ligands. 4. Product inhibition by inorganic phosphate is linear non-competitive with respect to phosphoenolpyruvate (Ki, slope = 22 mM and Ki, intercept = 54 mM) and substrate-linear competitive with respect to erythrose 4-phosphate (Ki, slope = 25 mM). 5. The enzyme has an activity optimum at pH 7.3 and a tryptophan inhibition optimum at pH 6.4, Trp 0.5 is 4 microM. Inhibition by tryptophan is non-competitive with respect to phosphoenolpyrovate and substrate-parabolic competitive with respect to erythrose 4-phosphate. 6. The role of the enzyme in metabolic regulation is discussed.     

Some properties of purified phosphoprotein phosphatases from rabbit liver.

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.     

Substrate-analogue-induced changes in the nickel-EPR spectrum of active methyl-coenzyme-M reductase from Methanobacterium thermoautotrophicum.

Methyl-coenzyme-M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M [2-(methylthio)ethanesulfonate] and 7-mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR-red1 and MCR-red2, probably derived from Ni(I). In the presence of the substrate methyl-coenzyme M, the rhombic MCR-red2 signal is quantitatively converted to the axial MCR-red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectrum of the enzyme. 3-Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki = 0.05 microM), converted the EPR signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal designated MCR-BrPrSO3. 3-Fluoropropanesulfonate (apparent Ki < 50 microM) and 3-iodopropanesulfonate (apparent Ki < 1 microM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I = 1/2, I = 3/2 and I = 5/2, respectively. This finding suggests that MCR-BrPrSO3 is not the result of a close halogen-Ni(I) interaction. 7-Bromoheptanoylthreonine phosphate (BrHpoThrP) (apparent Ki = 5 microM), which is an inhibitory substrate analogue of 7-mercaptoheptanoylthreonine phosphate, converted the signals MCR-red1 and MCR-red2 to a novel axial Ni(I) signal, MCR-BrHpoThrP, similar but not identical to MCR-BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to different enzyme/substrate and enzyme/inhibitor complexes.     

Effects of acyclovir and its metabolites on purine nucleoside phosphorylase

Acyclovir (9-(2-hydroxyethoxymethyl)guanine), the clinically useful antiherpetic agent, is an "acyclic" analogue of 2'-deoxyguanosine. Purine nucleoside phosphorylase partially purified from human erythrocytes did not catalyze detectable phosphorolysis of this drug or any of its metabolites (less than 0.07% of the rate with Guo). However, these compounds were competitive inhibitors of this enzyme with Ino as the variable substrate. Acyclovir per se was a relatively weak inhibitor. Its Ki value (91 microM) was much greater than that for its 8-hydroxy metabolite (Ki = 4.7 microM) but less than that for its carboxylic acid metabolite (9-carboxymethoxy-methylguanine) (K'i = 960 microM). The phosphorylated metabolites of acyclovir were more potent inhibitors than were their guanine nucleotide counterparts. At a phosphate concentration of 50 mM, the apparent Ki values for the mono- (120 microM), di- (0.51 microM), and tri (43 microM)-phosphate esters of acyclovir were 1/2, 1/1200, and 1/26 those for dGMP, dGDP, and dGTP, respectively. The concentration of phosphate did not markedly affect the Ki value of acyclovir but dramatically affected those of its phosphorylated metabolites and their nucleotide counterparts. Decreasing phosphate to a physiological concentration (1 mM) decreased the apparent Ki values for the mono-, di-, and triphosphate esters of acyclovir to 6.6, 0.0087, and 0.31 microM, respectively. Inhibition of the enzyme by acyclovir diphosphate was also influenced by pH. This metabolite of acyclovir is the most potent inhibitor of purine nucleoside phosphorylase reported to date. It has some features of a "multisubstrate" analogue inhibitor.     

Partial purification, substrate specificity and regulation of alpha-L-glycerolphosphate dehydrogenase from Saccharomyces carlsbergensis.

alpha-L-Glycerolphosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) from Saccharomyces carlsbergensis was purified 400-fold. The enzyme preparation is free of interfering activities, such as glyceraldehyde phosphate dehydrogenase, alcohol dehydrogenase, triose phosphate isomerase and glycerolphosphatase. At pH 7.0 it is specific for NADH (Km = 0.027 mM with 0.8 mM dihydroxyacetone phosphate) and dihydroxyacetone phosphate (Km = 0.2 mM with 0.2 mM NADH). Between pH 5.0 and 6.0 the enzyme functions with NADPH, but only at 7% of the rate with NADH. Various anions (I- greater than SO42- greater than Br- greater than Cl-) act as inhibitors competing with the substrate dihydroxyacetone phosphate. Inorganic phosphate (Ki = 0.1 mM), pyrophosphate and arsenate are strong inhibitors. The nucleotides ATP and ADP are also inhibitory, but their action seems to be of the same type as the general anion competition (Ki = 0.73 mM for ATP). The results are consistent with the notion that the enzyme may regulate the redox potential of the NAD+/NADH couple during fermentation.     

Solvent effects on substrate and phosphate interactions with the (Na+ + K+)-ATPase

(Na+ + K+)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10-30% (v/v) dimethyl sulfoxide (Me2SO) and ethylene glycol (Et(OH)2); moreover, Me2SO produced a pattern of uncompetitive inhibition toward ATP. However, K+-nitrophenylphosphatase activity was stimulated by 10-20% Me2SO and Et(OH)2 but was inhibited by 30-50%. Me2SO decreased the Km for this substrate but had little effect on the Vmax below 30% (at which concentration Vmax was then reduced). Me2SO also reduced the Ki for Pi and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the Ki for ATP, CTP and 2-O-methylfluorescein phosphate as competitors. Me2SO inhibited K+-acetylphosphatase activity, although it also reduced the Km for that substrate. Finally, Me2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E1P to E2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E2P intermediate, through differential solvation of reactants and products, and thereby inhibiting the (Na+ + K+)-ATPase reaction and acting as a dead-end inhibitor to produce the pattern of uncompetitive inhibition; inhibiting the K+-acetylphosphatase reaction that also passes through an E2P intermediate; but not inhibiting (at lower Me2SO concentrations) the K+-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of Pi and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the Km for nitrophenyl phosphate and acetyl phosphate, the Ki for Pi and acetyl phosphate in the K+-nitrophenylphosphatase reaction, accelerating inactivation by fluoride and beryllium acting as phosphate analogs, and, at higher concentrations, inhibiting the K+-nitrophenylphosphatase reaction by stabilizing the non-covalent E2.P intermediate of that reaction. In addition, Me2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased Ki for ATP, CTP and 3-O-methylfluorescein phosphate.     

K(+)- and Mg2(+)-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase.     

Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.     

Role of phosphate on the ADP-induced hysteretic inhibition of mitochondrial adenosine 5'-triphosphatase. Effects of the natural protein inhibitor

Preincubation of F1-ATPase with ADP and Mg2+ leads to ADP binding at regulatory site inducing a hysteretic inhibition of ATP hydrolysis, i.e., an inhibition that slowly develops after Mg-ATP addition (Di Pietro, A., Penin, F., Godinot, C. and Gautheron, D.C. (1980) Biochemistry 19, 5671-5678). It is shown here that inorganic phosphate (Pi) together with ADP during preincubation abolishes the time-dependence of the inhibition after the addition of the substrate Mg-ATP. This preincubation in the presence of both Pi and ADP slowly leads to a conformation of the enzyme immediately inhibited after the addition of the substrate Mg-ATP. The Pi effect is half-maximal at 35 microM and pH 6.6, whereas a limited effect is induced at pH 8.0. The preincubation of F1-ATPase with Pi and ADP must last long enough (t1/2 = 5 min). The effects can be correlated to the amount of Pi bound to the enzyme, 1 mol Pi per mol (apparent KD of 33 microM) at saturation. Pi neither modifies the ADP binding nor the final level of the concomitant inhibition. When Pi is not present in the preincubation, the final stable rate of ADP-induced hysteretic inhibition is always reached when a near-constant amount of Pi has been generated during Mg-ATP hydrolysis. Kinetic experiments indicate that preincubation with ADP and Pi decreases both Vmax and Km which would favor a conformational change of the enzyme. Taking into account the Pi effects, a more precise model of hysteretic inhibition is proposed. The natural protein inhibitor IF1 efficiently prevents the binding of Pi produced by ATP hydrolysis indicating that the hysteretic inhibition and the IF1-dependent inhibition obey different mechanisms.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by vanadate

Green crab (Scylla serrata) alkaline phosphatase is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by vanadate has been studied. The time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the enzyme in the presence of different Na3VO4 concentrations showed that, at each Na3VO4 concentration, the rate decreased with increasing time until a straight line was approached, the slopes of the straight lines being the same for all concentrations. The results suggest that the inhibition of the enzyme by Na3VO4 is a slow, reversible reaction with fractional residual activity. The microscopic rate constants were determined for the reaction of the inhibitor with the enzyme. As compared with Na2HPO4 (Ki = 0.95 mM), Na2HAsO4 (Ki = 1.10 mM), and Na2WO4 (Ki = 1.55 mM), the results suggest that Na3VO4 (Ki = 0.135 mM) is a considerably more potent inhibitor than other inhibitors.     

Purine catabolism in man: inhibition of 5'-phosphomonesterase activities from placental microsomes.

The 5'-phosphomonoesterase activity of 5'-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.5) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5'-nucleotidase suggest a sequential mechanism of interaction between AMP nad MgCl2, with a Km of 14 and 3 muM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhititors when AMP was the substrate, with a Ki slope of 120 muM. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. These observations suggest that 5'-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5'-nucleotidase.     

Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)(2)SO(4) precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS-PAGE it was found to be a monomeric protein with molecular mass 72+/-2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 microM) and highest specificity constant (V(max)/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 microM and Ki=205 microM, respectively).     

The K+-translocating Kdp-ATPase from Escherichia coli. Purification, enzymatic properties and production of complex- and subunit-specific antisera

The Kdp system from Escherichia coli is a derepressible high-affinity K+-uptake ATPase. Its membrane-bound ATPase activity was approximately 50 mumol g-1 min-1. The Kdp-ATPase complex was purified from everted vesicles by solubilization with the nonionic detergent Aminoxid WS 35 followed by DEAE-Sepharose CL-6B chromatography at pH 7.5 and pH 6.4 and gel filtration on Fractogel TSK HW-65. The overall yield of activity was 6.5% and the purity at least 90%. The isolated KdpABC complex had a high affinity for its substrates K+ (Km app. = 10 microM) and Mg2+-ATP (Km = 80 microM) and a narrow substrate specificity. The ATPase activity was inhibited by vanadate (Ki = 1.5 microM), fluorescein isothiocyanate (Ki = 3.5 microM), N,N'-dicyclohexylcarbodiimide (Ki = 60 microM) and N-ethylmaleimide (Ki = 0.1 mM). The purification protocol was likewise applicable to the isolation of a KdpA mutant ATPase which in contrast to the wild-type enzyme exhibited an increased Km value for K+ of 6 mM and a 10-fold lowered sensitivity for vanadate. Starting from the purified Kdp complex the single subunits were obtained by gel filtration on Bio-Gel P-100 in the presence of SDS. Both the native Kdp-ATPase and the SDS-denatured polypeptides were used to raise polyclonal antibodies. The specificity of the antisera was established by immunoblot analysis. In functional inhibition studies the anti-KdpABC and anti-KdpB sera impaired ATPase activity in the membrane-bound as well as in the purified state of the enzyme. In contrast, the anti-KdpC serum did not inhibit enzyme activity.     

Heterogeneous hydrolysis of substoichiometric ATP by the F1-ATPase from Escherichia coli.

The hydrolysis of 0.3 microM [alpha,gamma-32P]ATP by 1 microM F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate. The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate. Under all conditions examined, only 10-20% of the [alpha,gamma-32P]ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP. These features differ from those observed with the mitochondrial F1-ATPase. The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate. Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D. and Senior, A.E. (1989) J. Biol. Chem. 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic. Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate. The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E. coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently.     

An angiotensin converting enzyme inhibitor is a tight-binding slow substrate of carboxypeptidase A

Carboxypeptidase A-catalyzed hydrolysis of peptides and depsipeptides is competitively inhibited by N-(1-carboxy-5-t-butyloxycarbonylaminopentyl)-L-phenylalanine (Boc-CA-Phe, Ki = 1.3 microM) and the angiotensin converting enzyme inhibitor, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine (Z-CA-Gly-Phe, Ki = 4.5 microM). The latter compound is actually a slow substrate of carboxypeptidase. Indirect observation of inhibitor binding by stopped-flow measurement of radiationless energy transfer between carboxypeptidase tryptophans and dansylated substrates reveals slow binding for both compounds. The visible absorption spectrum of the complex of cobalt(II)-substituted carboxypeptidase and Z-CA-Gly-Phe, which differs from the corresponding spectrum of the Boc-CA-Phe complex, is remarkable in its resemblance to the spectrum of the complex between Co(II)carboxypeptidase and a transient intermediate previously observed during hydrolysis of peptide substrates. The spectrum slowly changes to that of the free enzyme indicating hydrolysis. Chromatographic quantitation of substrate and products confirms that carboxypeptidase converts Z-CA-Gly-Phe to Z-CA-Gly and L-Phe with an apparent kcat of 0.02 s-1. Absorption spectroscopy indicates that the Z-CA-Gly-Phe-Co(II)carboxypeptidase spectrum is not that of bound products. Moreover, spectral titrations indicate that the products (both with spectral Ki values of about 3 mM), as well as D-Phe, compete for the same site on the enzyme.