Superactivity of human phosphoribosyl pyrophosphate synthetase due to altered regulation by nucleotide inhibitors and inorganic phosphate

Phosphoribosyl pyrophosphate (PPRibP) synthetase activity was studied in cultured fibroblasts and lymphoblasts from a male child (patient 2-A) in whom inherited purine nucleotide and uric acid overproduction are accompanied by neurological deficits. Chromatographed or partially purified preparations of the child's enzyme showed 5-6-fold increased inhibitory constants (I0.5) for the noncompetitive inhibitors GDP and 6-methylthioinosine monophosphate but normal responsiveness to the competitive inhibitors ADP and 2,3-diphosphoglycerate. Activation of the PPRibP synthetase of patient 2-A by Pi was also abnormal with 3-4-fold reduced apparent KD values for Pi. Superactivity of the PPRibP synthetase of this child thus appeared to result from a combination of regulatory defects; selective resistance to noncompetitive inhibitors and increased responsiveness to Pi activation. Selective growth of the patient's fibroblasts in medium containing 6-methylthioinosine confirmed the functional significance of the in vitro inhibitor resistance of the aberrant enzyme. Fibroblasts and lymphoblasts derived from patient 2-A showed increased concentrations and rates of generation of PPRibP as well as increased rates of the pathways of purine base salvage and purine nucleotide synthesis de novo. The magnitudes of these increases in the child's cells exceeded those in cells with catalytically superactive PPRibP synthetases. These alterations as well as the in vitro kinetic abnormalities in the patient 2-A enzyme were expressed to a reduced degree in fibroblasts from the child's affected mother, supporting the proposal that this woman is a heterozygous carrier for X-linked enzyme superactivity.     

In vitro and in vivo age-related modification of human erythrocyte phosphoribosyl pyrophosphate synthetase.

Upon storage, human erythrocyte phosphoribosyl pyrophosphate synthetase (PRibPP synthetase, EC 2.7.6.1) from normal individuals was found to undergo a spontaneous dissociation into active enzyme components of much smaller molecular mass (60 000--90 000). These modified forms of enzyme exhibit kinetic properties different from the original large molecular weight enzyme (over 200 000). The small active components can be reversibly associated to form larger molecules in the presence of purine ribonucleotides as well as phosphoribosyl pyrophosphate (PRibPP). ATP was found to be most effective in associating PRibPP synthetase, while guanylate nucleotides seem to have no effect. The large molecular weight components, once separated from the milieu, were not able to undergo further dissociation. Fresh or stored human white cell tissue homogenates were found to lack the low-molecular-weight enzyme under all our experimental conditions. A characteristic enzyme modification similar to that observed in stored erythrocyte was also noted in erythrocytes of increasing ages. The physiological significance of these findings to the regulatory function of PRibPP synthetase in purine metabolism in vivo is discussed.     

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate.

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Purine biosynthesis de novo in bovine retina: purification and characterization of amidophosphoribosyl transferase and phosphoribosyl pyrophosphate synthetase.

The ability of bovine retina to synthesize purines de novo is shown for the first time. Amidophosphoribosyl transferase (EC 2.4.2.14), the enzyme controlling the rate of the process, and phosphoribosyl pyrophosphate synthetase (EC 2.7.6.1), the enzyme regulating the intracellular contents of phosphoribosyl pyrophosphate (PRPP), were purified and characterized. The molecular masses of the enzyme subunits are similar to those of the purified enzyme from the liver. The molecular masses of amidophosphoribosyl transferase, PRPP synthetase catalytic subunit, and two PRPP synthetase-associated proteins are 50, 34, 39, and 41 kD, respectively. The apparent Km values of the enzymes and coenzymes are similar to those of the purified enzymes from the liver. For amidophosphoribosyl transferase, the apparent Km for Gln and PRPP are 0.75 +/- 0.05 and 0.66 +/- 0.09 mM, respectively (the corresponding Vmax values are 59 +/- 3 and 136 +/- 12 nmoles PPi/min per mg protein). For PRPP synthetase, the apparent Km for ribose-5-phosphate and ATP are 37.9 +/- 0.5 and 53 +/- 7 microM, respectively (the corresponding Vmax values are 61 +/- 4 and 52 +/- 3 nmoles PRPP/min per mg protein). The sensitivity of the retinal PRPP synthetase to inhibition by ADP and AMP was significantly lower than that of the enzyme from the liver.     

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.     

Mechanisms of product feedback regulation and drug resistance in cytidine triphosphate synthetases from the structure of a CTP-inhibited complex

Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447-6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface.     

A direct, stimulating effect of cyclic GMP on purified phosphoribosyl pyrophosphate synthetase and its antagonism by cyclic AMP

The activity of phosphoribosyl pyrophosphate synthetase, purified from a line of rat hepatoma cells in continuous culture, is maximally stimulated (2–4 fold) by less than 10^?7M cyclic GMP. Half maximal stimulation occurs at 2 × 10^?9M. Cyclic GMP stimulates phosphoribosyl pyrophosphate synthetase by decreasing the Km of the enzyme for ATP from 50 μM to 10 μM without affecting the Vmax; it has no effect on the Km for ribose 5-phosphate, the other substrate. Cyclic AMP alone has no effect on the enzyme activity, but at micromolar concentrations it antagonizes the stimulation by cyclic GMP. GMP, GDP, and GTP do not stimulate enzyme activity; and AMP and ADP at micromolar concentrations do not antagonize the effect of cyclic GMP.There is no detectable cyclic nucleotide-activated protein kinase in the enzyme preparation. Cyclic GMP significantly stabilizes the enzyme to heat inactivation. We conclude that cyclic GMP binds directly to the enzyme in an allosteric fashion, causing it to have an increased affinity for one of its substrates, and that cyclic AMP directly antagonizes this effect.     

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon.

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.     

Feedforward inhibition in biosynthetic pathways: inhibition of the aminoacyl-tRNA synthetase by the penultimate product.

Inhibition of the aminoacyl-tRNA synthetase by the penultimate product of a pathway for the biosynthesis of an amino acid has been reported for several pathways in many different types of organisms. A regulatory role for this mechanism often has been suggested, although there is some conflicting experimental evidence. The significance of such feedforward inhibition is examined here by mathematical analysis. The techniques that have proved successful in the analysis of control by feedback inhibition—showing that the nearly universal pattern of end-product inhibition is an optimal design—indicate that feedforward inhibition by the penultimate product does not contribute significantly to the functional effectiveness of regulation at the level of enzymatic activity. Feedforward inhibition by the penultimate product may have no physiological role, or it may be involved in differential signalling of intra- and extracellular changes and/or in directing the metabolic flow in branched pathways. These possibilities are discussed in light of analytical results presented in this paper and published experimental evidence.     

Interrelated regulation of sulphur-containing amino-acid biosynthetic enzymes and folate-metabolizing enzymes in Aspergillus nidulans

In Aspergillus nidulans homocysteine can be metabolized both to cysteine and methionine. Mutants impaired in the main pathway of cysteine synthesis or in the sulphate assimilation pathway show a low pool of glutathione and elevated levels of homocysteine synthase and of the homocysteine-to-cysteine pathway enzymes. On the other hand, the level of methionine synthase and other enzymes of folate metabolism is depressed in these mutants. This anticoordinated regulation provides a mechanism controlling the partition of homocysteine between the two diverging pathways. Homocysteine synthase was found derepressed, along with folate enzymes, in a strain carrying a mutation which suppresses mutations in metA, metB and metG genes. These results indicate that homocysteine synthase can be regarded as the enzyme of an alternative pathway of methionine synthesis and strongly suggest that the regulatory mechanisms governing sulphur-containing amino acid and folate metabolisms are interrelated.     

Rat liver phosphoribosyl pyrophosphate synthetase: existence of the purified enzyme as heterogeneous aggregates and identification of the catalytic subunit

Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.     

Rhythms in glutamine synthetase activity, energy charge, and glutamine in sunflower roots

Roots of sunflower plants (Helianthus annuus L. var. Mammoth Russian) subjected to L12:D12, L18:D6, and L12:D12 followed by continuous light all display rhythms of about 12 hours for glutamine synthetase (GS) activity (transferase reaction) with one peak in the `light phase' and one in the `dark phase.' Root energy charge (EC = ATP+½ADP/ATP+ADP+AMP) is directly correlated with GS, but the GS rhythm is better explained as the result of a rhythmic adenine nucleotide ratio (ATP/ADP+AMP) that regulates enzyme activity through allosteric modification. When L12:D12 plants are subjected to free-running conditions in continuous darkness, only diurnal rhythms for GS and EC, with peaks in the dark phase, remain. The 12-hour root rhythms for GS and EC appear to be composed of two alternating rhythms, one a diurnal, light-dependent, incompletely circadian light phase rhythm and the other a light-independent, circadian dark phase rhythm.

Only glutamine, of the root amino acids, displays cyclical changes in concentration, maintaining under all conditions a 12-hour rhythm that is consistently synchronized with, but nearly always inversely correlated with, GS and EC rhythms.