REM sleep behavior disorder and degenerative dementia: an association likely reflecting Lewy body disease

It is commonly believed that MgATP2- is the substrate of F1-ATPases and ATP4- acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4- adds first and the binding of Mg2+ is dependent on the formation of the E x ATP4- complex. According to this ordered-binding model, solution formed MgATP2- is not recognized by the ATPase as a direct substrate, and the high-affinity binding of Mg2+ to the E x ATP4- complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1- ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP4-] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP4-] versus increasing [Mg2+]. For the ordered-binding alternative, a series of apparent dissociation constants were obtained for ATP4-(K(A)aPP), which decreased as [Mg2+] increased. From this family of K(A)aPP the true K(A) was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (K(B)aPP) and dependent on [ATP4-] as well as on the pH. The actual K(B) was established from a series of K(B)aPP by extrapolating to [ATP4-] = infinity and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2- (K(EAB)) of 0.0016 mM and for ATP4- (K(EA)) of 0.2 mM. Decision between the two models has far-reaching mechanistic implications. In the competitive inhibition model MgATP2- binds with high affinity, but Mg2+ cannot bind once the E x ATP4- complex is formed, while in the ordered-binding model binding of Mg2+ requires that ATP4- adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F-ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628] tend to favor the ordered-binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered-binding model gains more experimental support, a revision of the current concepts of unisite catalysis and negative cooperativity of nucleotide binding will be necessary.     

Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1

The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibition of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of MgATP by CF1-epsilon that contains tightly bound ADP, but no bound Mg2+, is initially rapid and decreases within about 1 min to a steady-state rate. The bound MgADP content of CF1-epsilon was varied. The initial fast phase of MgATP hydrolysis is eliminated when the molar ratio of MgADP to CF1-epsilon approaches 2. Loosely bound Mg2+ also affects the initial kinetics of the enzyme that contains bound MgADP. At molar ratios of bound MgADP to enzyme in excess of 1, the initial ATPase activity was low and reached the steady state after about 30 s. Free Mg2+ in the assay mix also inhibited steady-state ATP hydrolysis by all forms of the enzyme. The results are consistent with a model in which two Mg2+ bind cooperatively, probably to the dissociable nucleotide-binding sites on CF1-epsilon. Thus, four different nucleotide-binding sites may be involved in the inhibition of the MgATPase activity of CF1-epsilon. Three of these sites are potentially catalytic, and the fourth may be regulatory. The exchange of bound trinitrophenyl-ADP induced by the addition of MgATP or CaATP was found to be fast enough for the site to be involved in catalysis.     

Neither human immunodeficiency virus-1 (HIV-1) nor HIV-2 infects most-primitive human hematopoietic stem cells as assessed in long-term bone marrow cultures

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by alpha-oxidation of phenylacetyl-CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate-oxidoreductase to benzoyl-CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate:acceptor oxidoreductase was purified and studied. The oxygen-sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an alpha2 beta2 gamma2 delta2 epsilon2 composition. Phenylglyoxylate:acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron-sulfur protein and contained 35 +/- 4 mol Fe, 36 +/- 4 mol acid-labile sulfur, and 1.1 +/- 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 microM) and coenzyme A (Km 55 microM); 2-oxoisovalerate was oxidized with 15% of the rate. The turnover number with benzyl viologen at 37 degrees C was 46 s(-1) at the optimal pH of 8. The enzyme catalyzed a NAD(P)H:viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition alpha2 beta2 gamma2 delta2, as reported for archaeal 2-oxoacid:ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an epsilon2 unit that catalyzes the transfer of electrons from a small ferredoxin-like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin:NAD(P)+-oxidoreductase.     

Affinity labeling of rat liver carbamyl phosphate synthetase I by 5'-p-fluorosulfonylbenzoyladenosine

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been used to study the interaction of MgATP with rat liver carbamyl phosphate synthetase I. Incubation of the enzyme with concentrations of FSBA as low as 0.025 mM produced considerable inactivation (41% at 120 min); identical rates and extents of reaction were produced by 0.5, 1, and 2 mM FSBA. Of the substrates for carbamyl phosphate synthetase I, only MgATP protected against FSBA inactivation. In the presence of a constant concentration of MgATP, increasing the FSBA concentration led to increased inhibition. Conversely, an increase in MgATP concentration led to decreased inhibition from a constant concentration of FSBA. Other nucleotide triphosphates provided no protection against FSBA inactivation. Addition of dithiothreitol to the FSBA-inactivated enzyme led to partial reactivation, suggesting that cysteine residue(s) were involved in the FSBA reaction. 5,5'-Dithiobis(2-nitrobenzoic acid) titration of the free sulfhydryl groups on the enzyme confirmed that cysteine residues were involved in reaction with FSBA; titration of the enzyme after incubation in the absence and presence of FSBA yielded values of 21 and 18(+/- 1), respectively. Binding studies with 5'-p-fluorosulfonylbenzoyl[2-3H]adenosine indicated that: 4 amino acid residues were involved in reaction with FSBA; 2 of these reaction sites were cysteine residues and 2 were noncysteine residues; MgATP protected one of the cysteine residues and one of the noncysteine residues from reaction with FSBA; the MgATP-protected noncysteine residue is essential for fully activity. These data strongly suggest that FSBA is an affinity label for two distinct MgATP sites on carbamyl phosphate synthetase I.     

Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA delta 3-delta 2-isomerase from Clostridium aminobutyricum

4-Hydroxybutyryl-CoA dehydratase, the key enzyme in the metabolism of gamma-aminobutyrate in Clostridium aminobutyricum, represents approximately 15-25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg-1. The dehydratase catalyses the reversible conversion of 4-hydroxybutyryl-CoA (Km = 50 microM) to crotonyl-CoA and possesses a probably intrinsic vinylacetyl-CoA delta 3-delta 2-isomerase with a specific activity of 223 nkat mg-1. The equilibrium of the reversible dehydration was determined from both sides as K = [crotonyl-CoA]/[4-hydroxybutyryl-CoA] = 4.2 +/- 0.3. Cyclopropylcarboxyl-CoA was not converted to crotonyl-CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2 +/- 0.2 mol FAD (64 +/- 5% oxidized), 16 +/- 0.8 mol Fe and 14.4 +/- 1.2 mol inorganic sulfur, which probably form iron-sulfur clusters. Exposure to air resulted initially in a slight activation followed by irreversible inactivation. Concomitantly the vinylacetyl-CoA delta-isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanoferrate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration a non activated C-H bond at C-3 of 4-hydroxybutyryl-CoA has to be cleaved. A putative mechanism for 4-hydroxybutyryl-CoA dehydratase is proposed in which this cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutyryl-CoA to 4-hydroxycrotonyl-CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Finally isomerisation yields crotonyl-CoA. 4-Hydroxybutyryl-CoA dehydratase is quite distinct from 3-hydroxyacyl-CoA dehydratase (crotonase) and 2-hydroxyacyl-CoA dehydratases. Contrary to the latter enzyme [e.g. (R)-lactyl-CoA dehydratase and (R)-2-hydroxyglutaryl-CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C-H bond at C-3, 4-hydroxybutyryl-CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4-hydroxybutyryl-CoA dehydratase is not inactivated by oxidants such as 5 mM 4-nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.     

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and 相似文献   

MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic beta cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The beta cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.     

Differences in the magnesium dependences of the class I and class II aminoacyl-tRNA synthetases from Escherichia coli

The magnesium dependences of the ATP/PPi exchange and tRNA aminoacylation of reactions were measured for six aminoacyl-tRNA synthetases (isoleucyl-, tyrosyl- and arginyl-tRNA synthetases from class I, and histidyl-, lysyl- and phenylalanyl-tRNA synthetases from class II). The measured values were subjected to best-fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg2+ and to optimize the sets of the kinetic constants. The following four dependences were observed: the class II synthetases require three Mg2+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mg2+ (in MgATP); in class II synthetases both MgPPi and Mg2PPi participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl-tRNA synthetase from class I also shows a better fit if also Mg2PPi reacts, but in the isoleucyl- and tyrosyl-tRNA synthetases only MgPPi but not Mg2PPi is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA-bound Mg2+ and spermidine, independent of the enzyme class. 1-4 Mg2+ or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg2+ from the product aminoacyl-tRNA essentially enhances the subsequent dissociation of the aminoacyl-tRNA from the enzyme. The binding of ATP to the E. aminoacyl-tRNA complex also speeds up the dissociation of the aminoacyl-tRNA from most of these enzymes.     

Purification and characterization of inorganic pyrophosphatase from Methanobacterium thermoautotrophicum (strain delta H)

Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the archaebacterium Methanobacterium thermoautotrophicum (strain delta H). The enzyme was purified 850-fold in three steps to electrophoretic homogeneity. The soluble pyrophosphatase consists of four identical subunits: the molecular mass of the native enzyme estimated by gel filtration was approx. 100 kDa and denaturing polyacrylamide gel electrophoresis gave a single band of 25 kDa. The enzyme also may occur as an active dimer formed by dissociation of the tetramer. The pyrophosphate showed an optimal activity at 70 degrees C and a pH of 7.7 (at 60 degrees C) and was not influenced by dithiothreitol, sodium dithionite or potassium chloride. The enzyme was very specific for pyrophosphate (PPi) and Mg2+. Magnesium could be partially replaced by Co2+ (15%). The reaction was inhibited for 60% by 1 mM Mn2+ in the presence of 24 mM Mg2+. In addition, the enzyme was inhibited by potassium fluoride (50% at 0.9 mM). Kinetic analysis revealed positive co-operativity for both Mg2+ and PPi with Hill coefficients of 3.3 and 2.0, respectively. Under the experimental conditions at which the enzyme was present as its dimer, the apparent Km of PPi and magnesium were determined and were approx. 0.16 mM and 4.9 mM, respectively; Vmax was estimated at about 570 U/mg.     

Effects of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin on nucleotide binding to the three F1-ATPase catalytic sites measured using specific tryptophan probes

Equilibrium nucleotide binding to the three catalytic sites of Escherichia coli F1-ATPase was measured in the presence of the inhibitors azide, dicyclohexylcarbodiimide, and aurovertin to elucidate mechanisms of inhibition. Fluorescence signals of beta-Trp-331 and beta-Trp-148 substituted in catalytic sites were used to determine nucleotide binding parameters. Azide brought about small decreases in Kd(MgATP) and Kd(MgADP). Notably, under MgATP hydrolysis conditions, it caused all enzyme molecules to assume a state with three catalytic site-bound MgATP and zero bound MgADP. These results rule out the idea that azide inhibits by "trapping" MgADP. Rather, azide blocks the step at which signal transmission between catalytic sites promotes multisite hydrolysis. Aurovertin bound with stoichiometry of 1.8 (mol/mol of F1) and allowed significant residual turnover. Cycling of the aurovertin-free beta-subunit catalytic site through three normal conformations was indicated by MgATP binding data. Aurovertin did not change the normal ratio of 1 bound MgATP/2 bound MgADP in catalytic sites. The results indicate that it acts to slow the switch of catalytic site affinities ("binding change step") subsequent to MgATP hydrolysis. Dicyclohexylcarbodiimide shifted the ratio of catalytic site-bound MgATP/MgADP from 1:2 to 1.6:1.4, without affecting Kd(MgATP) values. Like azide, it also appears to affect activity at the step after MgATP binding, in which signal transmission between catalytic sites promotes MgATP hydrolysis.     

A membrane-bound NAD(P)+-reducing hydrogenase provides reduced pyridine nucleotides during citrate fermentation by Klebsiella pneumoniae

During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H2/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO2. Upon addition of NiCl2 (36 microM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H2-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)+ was also achieved by the addition of formate. In crude extracts, the H2:NAD+ oxidoreductase activity was 0.13 micromol min-1 mg-1, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 micromol of NADPH formed min-1 mg-1 at pH 7.0 and 1.7 micromol of NADH formed min-1 mg-1 at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na+/H+ antiporter monensin, the H2-dependent reduction of NAD+ by membrane vesicles decreased only slightly (about 16%). The NADP+- or NAD+-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H2 as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H2:NAD(P)+ oxidoreductase that provides reducing equivalents for the synthesis of cell material.     

Crossbridge scheme and the kinetic constants of elementary steps deduced from chemically skinned papillary and trabecular muscles of the ferret

Elementary steps of the crossbridge cycle in chemically skinned ferret myocardium were investigated with sinusoidal analysis. The muscle preparations were activated at pCa 4.82 and an ionic strength of 200 mM, and the effects of the change in the MgATP (S) and phosphate (Pi) concentrations on three exponential processes were studied at 20 degrees C. Results are consistent with the following crossbridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and Det includes all detached states (MS and MDP) and weakly attached states (AMS and AMDP). From our studies, we obtained K1a = 0.99 mM-1 (MgATP association), k1b = 270 s-1 (ATP isomerization), k-1b = 280 s-1 (reverse isomerization), K1b = k1b/k-1b = 0.95, k2 = 48 s-1 (crossbridge detachment), k-2 = 14 s-1 (reverse detachment), K2 = 3.5, k4 = 11 s-1 (crossbridge attachment), k-4 = 107 s-1 (reverse attachment), K4 = 0.11, and K5 = 0.06 mM-1 (Pi association). K6 is the rate-limiting step, and it is the slowest forward reaction in the cycle, which results in the rigor-like AM state. K1a (MgATP binding) is four times that of rabbit psoas, and K5 (Pi binding) is 0.3 times that of psoas, demonstrating that crossbridges in myocardium bind MgATP more and Pi less than psoas. The rate constants of ATP isomerization (k1b, k-1b), crossbridge detachment (k2, k-2), and crossbridge attachment (k4) steps are generally an order of magnitude slower than rabbit psoas. The reverse attachment step (k-4) is similar to that in psoas, indicating that this step may occur irrespective of the myosin type and possibly spontaneously. The above scheme with the deduced kinetic constants predicts the following crossbridge distributions at 5 mM MgATP2- and 8 mM Pi:AM (3%), AM S (15%), AM*S (14%), Det (50%), AM*DP (6%), and AM*D (12%). The actual number of attached crossbridges was measured to be 51 +/- 4% by the stiffness ratio during activation and after rigor induction, and a strong correlation was seen with the prediction. Our results are consistent with the hypothesis that force generation occurs at the Det-->AM*DPi transition, and the same force is maintained after the release of Pi.     

The role of calcium on the activity of ERcalcistorin/protein-disulfide isomerase and the significance of the C-terminal and its calcium binding. A comparison with mammalian protein-disulfide isomerase

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI) shows a 55% identity with mammalian protein-disulfide isomerase (PDI) (Lucero, H. A., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112-23119) is a high capacity low affinity Ca2+-binding protein and behaves as a Ca2+ storage protein in the ER of a living cell (Lucero, H. A., Lebeche, D., and Kaminer, B. (1998) J. Biol. Chem. 273, 9857-9863). Here we show that recombinant ECaSt/PDI bound 26 mol of Ca2+/mol and a C-terminal truncated mutant bound 14 mol of Ca2+/mol, both with a Kd of 2.8 mM in 50 mM KCl and 5.2 mM in 150 mM KCl. The percentage reduction in Ca2+ binding in the mutant corresponded with the percentage reduction of deleted pairs of acidic residues, postulated low affinity Ca2+-binding sites. 5 mM Ca2+ moderately increased the PDI activity of both ECaSt/PDI and the C-terminal truncated mutant on reduced RNase and insulin. Surprisingly, ECaSt/PDI in the absence of Ca2+ prevented the spontaneous reactivation of reduced bovine pancreatic trypsin inhibitor. In the presence of 1-5 mM Ca2+ (or 10 microM polylysine) ECaSt/PDI augmented the bovine pancreatic trypsin inhibitor reactivation rate. In contrast, the C-terminal truncated ECaSt/PDI augmented rBPTI reactivation in the absence of Ca2+ and 1-5 mM Ca2+ further accelerated the reactivation rate, responses similar to those obtained with mammalian PDI.     

Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.     

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-

The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change. This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme. Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30%. The saturation curve for this compound is hyperbolic with a Kd of 6 microM. The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum. The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2. The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism. Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88. However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8%. Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex. The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes. The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2. These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E. coli, responsible for the inhibition of the enzyme activity by this ligand.     

Thrombin-like enzyme from Lachesis muta muta venom: isolation and topographical analysis of its active site structure by means of the binding of amidines and guanidines as competitive inhibitors

A serine protease enzyme was purified from Lachesis muta muta venom, with 40% yield, by gel filtration on Sephadex G-100 and affinity chromatography on Sepharose-agmatin. Homogeneity of the enzyme preparation was demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the enzyme had a relative mol. wt of 45,000. The molar extinction coefficient at 280 nm was 62,127 (M x cm)-1. The enzyme hydrolysed Bz-Arg-Nan with Ks = 0.233 +/- 0.08 mM and kcat = 2.80 +/- 0.07 sec-1. All the amidines and guanidines tested for their inhibitory effect on thrombin-like enzyme behaved as competitive inhibitors of the enzyme with Ki values in the range 6.2 microM to 42.3 mM for amidines and 0.19 mM to 9.31 mM for guanidines. Dissociation constant values were analyzed in terms of the binding of the inhibitors with the subsite S1, the specificity pocket of the enzyme, Ki values were discussed in accordance with those for trypsin inhibition. beta-Naphthamidine was the strongest inhibitor, while guanidine was the weakest. The differences among the Ki values were interpreted in terms of the shape of the enzyme active site. For meta- and para-substituted benzamidinium ions a good correlation was found between log l/Ki and sigma Hammett values of the substituents. The substituent effects in the pi-electrons of the benzamidine ring were considered in the frame of Hückel molecular orbital theory. A model for the binding of p-benzamidine derivatives with the primary specificity S1 subsite of the enzyme active site was proposed.     

Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur

Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 100 degrees C by the fermentation of carbohydrates yielding acetate, CO2, and H2 as the primary products. If elemental sulfur (S0) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). From the cytoplasm of this organism, we have now purified an enzyme, sulfide dehydrogenase (SuDH), which catalyzes the reduction of polysulfide to H2S with NADPH as the electron donor. SuDH is a heterodimer with subunits of 52,000 and 29,000 Da. SuDH contains flavin and approximately 11 iron and 6 acid-labile sulfide atoms per mol, but no other metals were detected. Analysis of the enzyme by electron paramagnetic resonance spectroscopy indicated the presence of four iron-sulfur centers, one of which was specifically reduced by NADPH. SuDH has a half-life at 95 degrees C of about 12 h and shows a 50% increase in activity after 12 h at 82 degrees C. The pure enzyme has a specific activity of 7 mumol of H2S produced.min-1.mg of protein-1 at 80 degrees C with polysulfide (1.2 mM) and NADPH (0.4 mM) as substrates. The apparent Km values were 1.25 mM and 11 microM, respectively. NADH was not utilized as an electron donor for polysulfide reduction. P. furiosus rubredoxin (K(m) = 1.6 microM) also functioned as an electron acceptor for SuDH, and SuDH catalyzed the reduction of NADP with reduced P. furiosus ferredoxin (K(m) = 0.7 microM) as an electron donor. The multiple activities of SuDH and its proposed role in the metabolism of S(o) and polysulfide are discussed.     

Substrate and cofactor reactivity of a carbon monoxide dehydrogenase-corrinoid enzyme complex: stepwise reduction of iron-sulfur and corrinoid centers, the corrinoid Co2+/1+ redox midpoint potential, and overall synthesis of acetyl-CoA

Cleavage of the acetyl carbon-carbon bond of acetyl-CoA in Methanosarcina barkeri is catalyzed by a high molecular mass multienzyme complex. The complex contains a corrinoid protein and carbon monoxide dehydrogenase and requires tetrahydrosarcinapterin (H4SPt) as methyl group acceptor. Reactions of the enzyme complex with carbon monoxide and with the methyl group donor N5-methyltetrahydrosarcinapterin (CH3-H4SPt) have been analyzed by UV-visible spectroscopy. Reduction of the enzyme complex by CO occurred in two steps. In the first step, difference spectra exhibited peaks of maximal absorbance decrease at 426 nm (major) and 324 nm (minor), characteristic of Fe-S cluster reduction. In the second step, corrinoid reduction to the Co1+ level was indicated by a prominent peak of increased absorbance at 394 nm. Spectrophotometric analyses of the corrinoid redox state were performed on the intact complex at potentials poised by equilibration with gas mixtures containing different [CO2]/[CO] ratios or by variation of the [H+]/[H2] ratio. The corrinoid Co2+/1+ midpoint potential was -426 mV (+/- 4 mV, n = 1.16 electrons, 24 degrees C), independent of pH (pH 6.4-8.0). The results indicated a significant fraction of Co1+ corrinoid at potentials existing in vivo. The reduced corrinoid reacted very rapidly with CH3-H4SPt. Reaction with methyl iodide was slow, and methylation by S-adenosylmethionine was not observed. Tne rate of methyl group transfer from CH3-H4SPt greatly exceeded the rate of CO reduction of enzyme centers. The enzyme complex catalyzed efficient synthesis of acetyl-CoA from coenzyme A, CO, and CH3-H4SPt. During acetyl-CoA synthesis, demethylation of CH3-H4SPt was monitored by the absorbance increase at 312 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dephosphorylation activates the purified plant plasma membrane H+-ATPase--possible function of phosphothreonine residues in a mechanism not involving the regulatory C-terminal domain of the enzyme

The plasma membrane H+-ATPase was purified from tobacco cells (line BY-2). After solubilization by lysophosphatidylcholine followed by separation on a glycerol gradient, a fraction with a high specific activity of 9 micromol ATP x min(-1) x mg protein(-1) was obtained, in which the H+-ATPase polypeptide represented at least 80% of the protein. The incubation of this fraction in the presence of alkaline phosphatase increased H+-ATPase activity by 40%, in a manner consistent with dephosphorylation of the enzyme itself. The hydrolytic activity of the solubilized enzyme and its proton translocating activity, after reconstitution into proteoliposomes, were stimulated to the same extent. Alkaline phosphatase treatment was also accompanied by a 92% decrease in the H+-ATPase phosphothreonine content, whereas the phosphoserine residues were almost unaffected. The dephosphorylation induced a slight decrease of the affinity of the enzyme towards ATP. The purified enzyme was not activated by lysophosphatidylcholine addition nor by trypsin-mediated proteolysis, two treatments reported to release the inhibitory control by the C-terminal domain of the H+-ATPase and to increase the affinity of the enzyme towards ATP. Based on these results, the regulatory phosphorylation evoked by alkaline phosphatase most likely differs from the autoinhibitory control of the H+-ATPase by its C-terminal domain.