Mechanism of action of clostridial glycine reductase: isolation and characterization of a covalent acetyl enzyme intermediate

Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + Pi + 2e(-)----acetyl phosphate + NH4+. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. We now demonstrate that protein C catalyzes exchange of [32P]Pi into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, we have isolated acetyl protein C and shown that it is qualitatively catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B [Arkowitz, R. A., & Abeles, R. H. (1990) J. Am. Chem. Soc. 112, 870-872]. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with Pi to give acetyl phosphate. When [14C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. At pH 11.5 radioactivity was released with t1/2 = 57 min, comparable to the hydrolysis rate of thioesters. Exposure of 4 N neutralized NH2OH resulted in the complete release of radioactivity. Treatment with KBH4 removes all the radioactivity associated with protein C, resulting in the formation of [14C]ethanol. We conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [3H]H2O into acetyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanistic and stereochemical studies on the glycine reductase of Clostridium sticklandii.

Clostridial glycine reductase multienzyme complex which catalyses the reaction: Glycine + ADP + Pi + 2H leads to Acetate + ATP + NH3 was solubilised and fractionated essentially according to the method of Stadtman [T.C. Stadtman (1970) Methods Enzymol. 17A, 956--966] into two components: protein A and 'glycine reductase' fraction. A reconstituted system obtained by combining the two components in the presence of dithiothreitol catalysed the conversion of glycine into acetate concomitant with the phosphorylation of ADP to ATP. Using the reconstituted system, in which the unwanted enzyme activity catalyzing an exchange of the alpha hydrogen atoms of glycine with the protons of the medium had been greatly reduced, it was found that the conversion of (2RS)-[2-14C, 2-3H1]glycine (3H/14C = 7.16) into acetate (3H/14C = 7.03) was attended by the retention of both the C-2 hydrogen atoms of glycine. Conversion of (2S)-[2-2H1, 2-3H1]glycine and (2R)-[2-2H1, 2-3H1]glycine by the reconstituted system gave (2S)-acetate and (2R)-acetate respectively showing that the reductive deamination of glycine occurs through an inversion of configuration. The cumulative information available on the glycine reductase reaction is embodied in a hypothetical mechanism of action for the enzyme.     

Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima

Phosphate acetyltransferase (PTA) and acetate kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.     

Glycine reductase protein C. Properties and characterization of its role in the reductive cleavage of Se-carboxymethyl-selenoprotein A

The clostridial glycine reductase complex catalyzes the reductive deamination of glycine in an energy-conserving process that results in the esterification of orthophosphate. The complex consists of three protein components: selenoprotein A; protein B, a carbonyl group protein; and protein C, a sulfhydryl protein. The protein C component also catalyzes the arsenate-dependent decomposition of acetyl phosphate. Reaction of protein C with iodoacetate inhibits its ability to decompose acetyl phosphate, but this inactivation of the enzyme by alkylation is prevented in the presence of the substrate indicating the formation of an unreactive enzyme-bound acetylthiol ester. The Se-carboxy-methylselenocysteine residue of the selenoprotein A component of glycine reductase was generated by selective alkylation of the ionized selenol group at pH 6 with [14C]bromoacetate. Using this pure alkylated selenoprotein A as substrate, it was shown that protein C catalyzes the conversion of the [14C]carboxymethyl group, in selenoether linkage to protein A, to [14C]acetate in the presence of arsenate, dithiothreitol, and Mg2+. A procedure using hydrophobic chromatographic matrices was developed for the large scale isolation of protein C, and a number of the properties of the enzyme were determined.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra.

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:     

Mechanism of acetate synthesis from CO2 by Clostridium acidiurici.

Total synthesis of acetate from CO2 by Clostridium acidiurici during fermentations of hypoxanthine has been shown to involve synthesis of glycine from methylenetetrahydrofolate, CO2, and NH3. The glycine is converted to serine by the addition of methylenetetrahydrofolate, and the resulting serine is converted to pyruvate, which is decarboxylated to form acetate. Since CO2 is converted to methylenetetrahydrofolate, both carbons of the acetate are derived from CO2. The evidence supporting this pathway is based on (i) the demonstration that glycine decarboxylase is present in C. acidiurici, (ii) the fact that glycine is synthesized by crude extracts at a rate which is rapid enough to account for the in vivo synthesis of acetate from CO2, (iii) the fact that methylenetetrahydrofolate is an intermediate in the formation of both carbons of acetate from CO2, and (iv) the fact that the alpha carbon of glycine is the source of the carboxyl group of acetate. Evidence is presented that this synthesis of acetate does not involve carboxylation of a methyl corrinoid enzyme such as occurs in Clostridium thermoaceticum and Clostridium formicoaceticum. Thus, there are two different mechanisms for the total synthesis of acetate from CO2 by clostridia.     

Effect of zinc ion on the interaction of some amino acid compounds of copper(II) with hydrogen peroxide.

Reaction of elemental copper and zinc powder mixtures with glycine (NH2.CH2COOH; HA) or aspartic acid (NH2CHCOOHCH2COOH; H2B) (in 1:1:2 ratio, respectively) in the presence of excess hydrogen peroxide (H2O2) at 50 degrees C, results in the formation of a new mixed metal peroxy carbonate compound corresponding to formula [Cu(Zn)2(O2(2-) (CO3)2(H2O)4], while the same reaction with elemental copper powder alone yields merely peroxy amino acid compounds having the formula [Cu(O2(2-)) (HA)2(H2O)] and [Cu(O2(2-)) (H2B) (H2O)2] for glycine and aspartic acid, respectively. These compounds have been characterized by elemental analysis, ESR, and electronic and IR spectra. It is interesting to note that both amino acids are converted to carbonate in the presence of zinc alone. A method analogous to that described above, for the reaction of elemental copper, zinc powder mixtures with succinic acid [(CH2COOH)2] or acetic acid (CH3COOH) in excess H2O2, on the other hand, gave a product essentially comprising copper succinate or acetate, respectively. These observations suggest an interesting and perhaps important phenomenon by which only the simple amino acids such as glycine and aspartic acid are converted to carbonates while their corresponding carboxylic acids form only their respective salts.     

Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and coupling with aerobic utilization of lactate

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H2O2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147:2469-2477, 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H2O2-producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H2O2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H2O2 production in S. pyogenes. Of the other two potential H2O2-producing enzymes of this bacterium, NADH and alpha-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H2O2-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H2O2. Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H2O2-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.     

Acetate kinase from Veillonella alcalescens. Regulation of enzyme activity by succinate and substrates.

Acetate kinase of Veillonella alcalescens has been shown to be highly regulated enzyme exhibiting two levels of control: the requirement for succinate as a heterotropic allosteric effector, and cooperative binding at the substrate level. Succinate addition was necessary for enzymatic activity in both the direction of acyl phosphate synthesis and that of ATP synthesis. Control at the substrate level was apparent in the cooperative binding (Hill coefficients of 2) of acetyl phosphate, ATP, and ADP. Typical Michaelis kinetic data were observed for succinate (Ka = 20 mM for acetyl phosphate synthesis, 0.4 mM for ATP synthesis), acetate, and propionate. The primary effect of succinate was to increase the apparent Vmax of the enzymatic reaction for the variable substrates, ATP, ADP, and acetyl phosphate. The results are interpreted as evidence that, as a heterotropic effector of the acetate kinase reaction, succinate may regulate levels of propionyl-CoA (produced from propionyl phosphate by action of phosphotransacetylase), a compound required for the conversion of succinate to propionate. Acetase kinase has been shown to be a probable dimeric protein composed of two subunits of molecular weight 44,000 each.     

Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.     

Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.     

Cys359 of GrdD is the active-site thiol that catalyses the final step of acetyl phosphate formation by glycine reductase from Eubacterium acidaminophilum.

In the amino-acid-fermenting anaerobe Eubacterium acidaminophilum, acetyl phosphate is synthesized by protein C of glycine reductase from a selenoprotein A-bound carboxymethyl-selenoether. We investigated specific thiols present in protein C for responsibility for acetyl phosphate liberation. After cloning of the genes encoding the large and the small subunit (grdC1, grdD1), they were expressed separately in Escherichia coli and purified as Strep-tag proteins. GrdD was the only subunit that catalysed arsenate-dependent hydrolysis of acetyl phosphate (up to 274 U.mg-1), whereas GrdC was completely inactive. GrdD contained two cysteine residues that were exchanged by site-directed mutagenesis. The GrdD(C98S) mutant enzyme still catalysed the hydrolysis of acetyl phosphate, but the GrdD(C359A) mutant enzyme was completely inactive. Next, these thiols were analysed further by chemical modification. After iodoacetate treatment of GrdD, the enzyme activity was lost, but in the presence of acetyl phosphate enzyme activity was protected. Subsequently, the inactivated carboxymethylated enzyme and the protected enzyme were both denatured, and the remaining thiols were pyridylethylated. Peptides generated by proteolytic cleavage were separated and subjected to mass spectrometry. Cys98 was not accessible to carboxymethylation by iodoacetate in the native enzyme in the presence or absence of the substrate, but could be alkylated after denaturation. Cys359, in contrast, was protected from carboxymethylation in the presence of acetyl phosphate, but became accessible to pyridylethylation upon prior denaturation of the protein. This clearly confirmed the catalytic role of Cys359 as the active site thiol of GrdD responsible for liberation of acetyl phosphate.     

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.     

Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.     

Ubiquitin adenylate: structure and role in ubiquitin activation

The acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.     

Phosphatase activity of Na+/K+-ATPase. Enzyme conformations from ligands interactions and Rb occlusion experiments

The present work compares the effects of several ligands (phosphatase substrates, MgCl2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E2(Rb) occluded conformation of Na+/K+-ATPase. Cooling from 37 degrees C to 20 degrees C and 0 degrees C (hydrolysis experiments) or from 20 degrees C to 0 degrees C (occlusion experiments) had the following consequences: (i) dramatically reduced the Vmax for p-nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the Km for the substrates; (ii) led to a 5-fold drop in the Km for the inorganic phosphate-induced di-occlusion of E2(Rb); (iii) reduced the K0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p-nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E2(Rb) formation. At 20 degrees C, in the presence of 1 mM RbCl and no Mg2+, acetyl phosphate did not affect E2(Rb); with 3 mM MgCl2, acetyl phosphate stimulated a release of Rb from E2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the Km for iRb release was indistinguishable from the Km found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal hydrolysis was not different from 100%. These results indicate that although E2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E2 which has no K+ occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na+ + K+-dependent ATP hydrolysis; on the other hand, there are similarities with the reversible binding of inorganic phosphate in the presence of Mg2+ and K+ ions.     

Interactions of phosphate groups of ATP and Aspartyl phosphate with the sarcoplasmic reticulum Ca2+-ATPase: an FTIR study

Phosphate binding to the sarcoplasmic reticulum Ca2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([beta-18O2, betagamma-18O]ATP and [gamma-18O3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm(-1) are affected by labeling of the beta-phosphate, bands likely near 1154, and 1098-1089 cm(-1) are affected by gamma-phosphate labeling. The findings indicate that the strength of interactions of beta- and gamma- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm(-1), were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca2E1P phosphate less susceptible to attack by water.     

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.