pyrophosphate and acetyl-phosphate

ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires 6 phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleoside triphosphates, remains a mystery. Here, we show that the deep conservation of ATP might reflect its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate (AcP), which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that AcP can phosphorylate ADP to ATP at nearly 20% yield in water in the presence of Fe3+ ions. We then show that Fe3+ and AcP are surprisingly favoured. A wide range of prebiotically relevant ions and minerals failed to catalyse ADP phosphorylation. From a panel of prebiotic phosphorylating agents, only AcP, and to a lesser extent carbamoyl phosphate, showed any significant phosphorylating potential. Critically, AcP did not phosphorylate any other nucleoside diphosphate. We use these data, reaction kinetics, and molecular dynamic simulations to infer a possible mechanism. Our findings might suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.

Topics: Acetyl Coenzyme A; Adenosine Diphosphate; Adenosine Triphosphate; Carbamyl Phosphate; Diphosphates; Kinetics; Nucleosides; Organophosphates; Water

Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP(i))/inorganic phosphate (P(i)) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PP(i) formation but not in the direction of acetyl phosphate/P(i) formation suggests that the physiological direction of the reaction is toward acetate/PP(i) production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PP(i)-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.

Topics: Amino Acid Sequence; Binding Sites; Catalytic Domain; Diphosphates; Entamoeba histolytica; Molecular Sequence Data; Organophosphates; Phosphotransferases (Carboxyl Group Acceptor); Protozoan Proteins

Progress toward a protometabolism (the earliest energy storage networks) has been severely hindered by a shortage of driver reactions, which could have harnessed solar photons or coupled electron sources/sinks on the primordial Earth. Here, it is reported for the first time that thioacetate can be converted into a known metabolite, acetyl phosphate, by ultraviolet light and in aqueous solution at neutral pH. Of more compelling importance, the synthesis is catalyzed by uracil, which suggests that a genetic component may have also facilitated the emergence of metabolic pathways. The chemistry of acetyl phosphate has been extensively studied, and it is known to be a precursor of phosphate esters, pyrophosphate and possibly longer inorganic chains. Moreover, its bifunctional reactivity (as either an acetyl or phosphoryl donor) would have been integral for the first metabolic cycles.

Topics: Catalysis; Diphosphates; Hydrogen-Ion Concentration; Organophosphates; Photochemical Processes; Uracil

The formation of pyrophosphate as a result of nucleophilic attack by orthophosphate at the acylphosphate bond of acetyl phosphate was detectable in completely aqueous media, and was enhanced by dimethyl sulfoxide. The reaction had an absolute requirement for divalent cations, the rate constant of phosphorolysis being dependent on the species and concentration of cations as well as on temperature and pH. The amount of pyrophosphate formed depended on both the acetyl phosphate and orthophosphate concentrations. In purely aqueous media, phosphorolysis was barely detectable in the presence of Mg2+, and its rate increased 40-fold when Mg2+ was replaced by Ca2+ or Sr2+. In the presence of Mg2+ the rate of phosphorolysis increased 400-fold when 50 to 80% of the water was replaced by dimethyl sulfoxide. In the latter case, the rate also increased as the pH was raised from 4.0 to 9.0. The entropy of activation was large and negative in the presence of Mg2+ or Ca2+, indicating that the nucleophile is involved in the rate-limiting step of the reaction. Since this thermodynamic parameter became large and positive in the presence of Ca2+ when dimethyl sulfoxide was omitted, it is inferred that the transition state of the same reaction may be changed by the solvent composition and the solvation of reactants.

Topics: Cations, Divalent; Chemical Phenomena; Chemistry; Dimethyl Sulfoxide; Diphosphates; Organophosphates; Organophosphorus Compounds; Phosphates; Phosphorylation; Thermodynamics