Pyrophosphorylation of Ribose 5-Phosphate in the Enzymatic Synthesis of 5-Phosphorylribose 1-Pyrophosphate

Khorana, H. G.; Fernandes, J.F.; Kornberg, Arthur

doi:10.1016/s0021-9258(18)70517-6

Cited by 131 publications

(14 citation statements)

References 14 publications

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“…This method involves the intermediate information of PRPP followed by its nonenzymic decomposition in base as indicated in Scheme I. This decomposition of PRPP was originally reported by Khorana et al (1958), and we demonstrate here that the 8.7bridge oxygen is released quantitatively with the P¡. Since this P¡ contains all four 7-oxygens, it is possible to simultaneously follow both PIX and washout of the 7-nonbridge oxygens during ATP *=* HOH exchange on the same ATP sample.…”

supporting

confidence: 61%

Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Dale¹,

Hackney²

1987

Biochemistry

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A method for analysis of positional isotope exchange (PIX) during ATP in equilibrium with HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the beta P-O gamma P bond. This cleavage yields Pi derived from the gamma-phosphoryl of ATP that contains all four of the gamma oxygens. Both PIX between the beta,gamma-bridge and beta-nonbridge positions and washout of the gamma-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17O at the beta-nonbridge positions and 18O at the beta,gamma-bridge and gamma-nonbridge positions. Application of this method to ATP in equilibrium with HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of gamma-nonbridge oxygens in the virtual absence of PIX. At 25 degrees C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s-1, which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the beta-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 degrees C, this fraction corresponds to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. [Geeves, M. A., Webb, M. R., Midelfort, C. F., & Trentham, D. R. (1980) Biochemistry 19, 4748-4754] with subfragment 1.

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supporting

confidence: 61%

Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Dale¹,

Hackney²

1987

Biochemistry

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“…The enzyme 5-phospho-D-ribosyl R-1-diphosphate (PRPP) 1 synthetase (EC 2.7.6.1) catalyzes the reaction Rib-5-P + ATP f PRPP + AMP, which proceeds by attack of the 1-hydroxyl of Rib-5-P on the β-phosphoryl of ATP resulting in the transfer of the β,γ-diphosphoryl moiety to Rib-5-P (Khorana et al, 1958;Miller et al, 1975). PRPP is a precursor of purine, pyrimidine, and pyridine nucleotides and in plants and microorganisms of the amino acids tryptophan and histidine Hove-Jensen, 1988, 1989.…”

mentioning

confidence: 99%

Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

Willemoës

Hove‐Jensen

1997

Biochemistry

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The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP and (+)-1-alpha,2-alpha,3-alpha-trihydroxy-4-beta-cyclopentanemethanol 5-phosphate, respectively, the binding of Mg2+ and the substrates were determined to occur via a steady state ordered mechanism in which Mg2+ binds to the enzyme first and ribose 5-phosphate binds last. Mg2+ binding to the enzyme prior to the binding of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands Mg x ATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate. Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by Mg x ADP indicated that Mg x ADP binding to the allosteric site may occur in competition with enzyme bound Mg2+. Ligand binding studies showed that 1 mol of Mg x ATP was bound per mol of phosphoribosyl diphosphate synthetase subunit, which indicated that the allosteric sites of the multimeric enzyme were not made up by inactive catalytic sites.

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“…The enzyme 5-phospho-D-ribosyl R-1-diphosphate (PRPP) 1 synthetase (EC 2.7.6.1) catalyzes the reaction Rib-5-P + ATP f PRPP + 5′-AMP by attack of the 1-hydroxyl of Rib-5-P on the β-phosphate of ATP, resulting in transfer of the β,γdiphosphoryl moiety to Rib-5-P (Khorana et al, 1958). The Salmonella typhimurium PRPP synthetase reaction occurs by a sequential, ordered mechanism with ATP binding to the enzyme first and PRPP dissociating last (Switzer, 1971).…”

mentioning

confidence: 99%

Effects of Mutagenesis of Aspartic Acid Residues in the Putative Phosphoribosyl Diphosphate Binding Site of Escherichia coli Phosphoribosyl Diphosphate Synthetase on Metal Ion Specificity and Ribose 5-Phosphate Binding

1996

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The three conserved aspartic acid residues of the 5-phospho-D-ribosyl alpha-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg2+ or Mn2+, respectively, whereas Vapp was similar to that of the wild-type and KM for Rib-5-P was increased 4-fold in the presence of Cd2+. The changes in KM for ribose 5-phosphate and Vapp of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis.

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Pyrophosphorylation of Ribose 5-Phosphate in the Enzymatic Synthesis of 5-Phosphorylribose 1-Pyrophosphate

Cited by 131 publications

References 14 publications

Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Analysis of positional isotope exchange in ATP by cleavage of the .beta.P-O.gamma.P bond. Demonstration of negligible positional isotope exchange by myosin

Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

Effects of Mutagenesis of Aspartic Acid Residues in the Putative Phosphoribosyl Diphosphate Binding Site of Escherichia coli Phosphoribosyl Diphosphate Synthetase on Metal Ion Specificity and Ribose 5-Phosphate Binding

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