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All organisms, with the exception of protozoa, synthesize purines. Our knowledge of the purine biosynthetic reaction sequence largely evolved from studies in birds. PRPP availability determines the rate of purine biosynthesis in cells. Gly, Asp, and Gln are amino acids used in purine biosynthesis. All intermediates involved in purine biosynthesis are constructed on a ribose 5-phosphate “scaffold.” AMP and GMP are synthesized from IMP, the “parent” nucleotide. Regulation of purine biosynthesis occurs at the beginning and end of the pathway.

Overview • An insulin-independent GLUT 1 transporter helps to facilitate glucose entry into erythrocytes.

Publisher Summary This chapter describes the assay methodology of phosphoribosylpyrophosphate synthetase (PRPP) (ribose-5-phosphate pyrophosphokinase) from Salmonella typhimurium . PRPP synthetase can be conveniently assayed in most tissues by measuring the transfer of radioactivity from [γ- 32 P] adenosine triphosphate (ATP) to PRPP (and the products of the acid hydrolysis of PRPP) after the removal of unreacted ATP by charcoal adsorption. The procedure can be applied to crude cell extracts containing ATPase activities if the apparent activity of a control lacking ribose-5-phosphate is subtracted from each experimental measurement. The assay procedure can be used with a variety of crude cell extracts, so long as NaF is included and appropriate blanks are subtracted. The reaction of PRPP with orotate to form orotidylic acid and uridylic acid forms the basis of a spectrophotometric assay for PRPP synthetase, but this assay is not sufficiently sensitive for kinetic work. An alternative form of this assay in which release of 14 CO 2 from [7- 14 C] orotate is measured is also described in the chapter.

Publisher Summary This chapter describes the synthesis of ribulose 1,5-bisphosphate. The synthesis of ribulose 1,5-bisphosphate (RuBP) is accomplished by two routes: (1) glucose 6-phosphate and (2) adenosine monophosphate. These routes provide procedures suitable for preparing several hundred grams of RuBP and of the major intermediates lying between starting materials and this metabolite. The enzymes required are all used in an immobilized form. Both routes require ATP cofactor recycling and the procedure includes the details of an improved synthesis of the acetyl phosphate used as the ultimate phosphate donor for this recycling. The route based on glucose 6-phosphate (G-6-P) also illustrates a convenient procedure for the regeneration of nicotinamide adenine dinucleotide phosphate [NAD(P)] from nicotinamide adenine dinucleotide phosphate dehydrogenase [NAD(P)H] under anaerobic conditions, based on the conversion of α-ketoglutarate to glutamate. It is important to minimize the exposure of the RuBP to isomerizing conditions during and after preparation. The procedures for the preparation of RuBP are also summarized in the chapter. Glucose 6-phosphate is prepared using hexokinase-catalyzed phosphorylation and adenosine triphospahte (ATP) regeneration. Two methods employed for RuBP determination are the coupled-enzyme and radiometric method.

Publisher Summary This chapter discusses the preparation and analysis of ribose-5-phosphate. Two methods of preparation of ribose-5-phosphate are presented in this chapter. One of these is based on the hydrolysis of adenylic acid; the other involves the esterification of free ribose with ATP in the presence of ribokinase and is useful for the preparation of ribose-5-phosphate labeled with C 14 or p 32 . For hydrolysis of adenylic acid one gram of adenosine-5- phosphoric acid is dissolved in 10 ml of 1 N hydrochloric acid and heated for 30 minutes in a boiling water bath. The solution is cooled and centrifuged. The precipitate which forms when the extracts are combined is dissolved by the addition of water, and the solution is treated with of ethanol. The precipitate is collected by centrifugation and dried in vacuo water bath. In the second method, ribokinase is isolated and is assayed in a mixture containing 0.05 ml. of 0.02 M D-ribose, 0.05 ml. of 0.10 M MgCl 2 , 0.05 ml. of 0.045 M ATP, 0.50 ml. of 0.05 M Tris buffer, pH 7.5, and 0.05 to 0.20 ml. of ribokinase, incubated at 25 °.

Publisher Summary This chapter describes the assay method and properties of 5-phosphoribose pyrophosphokinase. The reaction is assayed by the convenient two-step procedure of Kornberg. In step I, the pyrophosphokinase reaction is carried out leading to the accumulation of 5-phosphoribosylpyrophosphate (PRPP), and in step II the accumulated PRPP is quantitatively utilized for the enzymatic conversion of orotate to a mixture of orotidylate and UMP. The removal of orotate in step II is determined spectrophotometrically. Alternatively, two other two-step procedures of greater sensitivity (but less convenience) are available. These involve the enzymatic conversion of C-adenine and 5-amino-4-imidazolecarboxamide to their respective ribotides. The assay method has been successfully applied to a wide variety of crude tissue extracts. The enzyme appears to be specific for ATP and ribose 5-phosphate. ADP, ITP, and UTP, at concentrations equal to that of the ATP used in the assay, either failed to replace ATP or showed less than 2% of the activity of ATP. Similarly, ribose 1-phosphate, 2-deoxyribose 5-phosphate, and glucose 6-phosphate failed to replace ribose 5-phosphate. 2-Deoxyribose 5-phosphate in the presence of an equimolar concentration of ribose 5-phosphate did not inhibit the formation of PRPP.

Publisher Summary This chapter discusses the preparation and purification of adenylic acid ribosidase. Two independent methods were used for the determination of AMP cleavage. The first procedure was based on the liberation of the free base, and the second on the appearance of reducing sugar. All the metals which activated purified 5′-AMP ribosidase stimulated the A. vinelandii myokinase activity. No means were found to avoid troublesome myokinase activity, except by selective destruction of this enzyme by prolonged heating. It was observed that Mn ++ markedly inhibits adenase, and this ion was useful for preventing the deamination of adenine to hypoxanthine, because the removal of adenase from 5′-AMP ribosidase was not complete. Starting with AMP and ATP, there was a disappearance of AMP with the appearance of equal amounts of reducing sugar and free base. It is observed that ATP was quantitatively recovered, indicating that this compound was not utilized during the course of the reaction.

SnRK1 of the SNF1/AMPK group of protein kinases is an important regulatory protein kinase in plants. SnRK1 was recently shown as a target of the sugar signal, trehalose 6-phosphate (T6P). Glucose 6-phosphate (G6P) can also inhibit SnRK1 and given the similarity in structure to T6P, we sought to establish if each could impart distinct inhibition of SnRK1. Other central metabolites, glucose 1-phosphate (G1P), fructose 6-phosphate and UDP-glucose were also tested, and additionally ribose 5-phosphate (R5P), recently reported to inhibit SnRK1 strongly in wheat grain tissue. For the metabolites that inhibited SnRK1, kinetic models show that T6P, G1P and G6P each provide distinct regulation (50% inhibition of SnRK1 at 5.4 μM, 480 μM, >1 mM, respectively). Strikingly, G1P in combination with T6P inhibited SnRK1 synergistically. R5P, in contrast to the other inhibitors, inhibited SnRK1 in green tissues only. We show that this is due to consumption of ATP in the assay mediated by phosphoribulokinase during conversion of R5P to ribulose-1,5-bisphosphate. The accompanying loss of ATP limits the activity of SnRK1 giving rise to an apparent inhibition of SnRK1. Inhibition of SnRK1 by R5P in wheat grain preparations can be explained by the presence of green pericarp tissue; this exposes an important caveat in the assessment of potential protein kinase inhibitors. Data provide further insight into the regulation of SnRK1 by metabolites.