Analogues of ribose 5-phosphate and 5-phosphoribosyl pyrophosphate. The preparation and properties of ribose 5-phosphorothioate and 5-phosphoribosyl 1-methylenediphosphonate

Murray, Andrew W.; Wong, Pak-Kin; Friedrichs, Beverly

doi:10.1042/bj1120741

Cited by 13 publications

(5 citation statements)

References 11 publications

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“…To further investigate the role of lysine residues in the mechanism of OPRTase, we examined a variety of possible analogs of pyrophosphate as alternative substrates and inhibitors. Previously, 5-phosphoribosyl 1 -0-( 1 -thiodiphosphate) (PRPPaS), 5-phosphoribosyl l-0-(2-thiodiphosphate) (PRPP/?S), and 5-phosphoribosyl-l-methylenediphosphonate (PRPCP) have been shown to act as alternative substrates for OPRTase (Murray et al, 1969;Smithers & O'Sullivan, 1984;McClard et al, 1984); and 5-phospho-2-deoxyribosyl-l-phosphonyldiphosphate is a good inhibitor of this enzyme (Witte & McClard, 1993).…”

Section: Resultsmentioning

confidence: 99%

Locations and Functional Roles of Conserved Lysine Residues in Salmonella typhimurium Orotate Phosphoribosyltransferase

Ozturk¹,

Dorfman²,

Scapin³

et al. 1995

Biochemistry

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Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in kcat. Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12-fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Locations and Functional Roles of Conserved Lysine Residues in Salmonella typhimurium Orotate Phosphoribosyltransferase

Ozturk¹,

Dorfman²,

Scapin³

et al. 1995

Biochemistry

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“…Reactions catalyzed by P-Rib-PP synthetase from S. typhimurium, leading to the synthesis of P-Rib-PP/JS and the diastereomers of P-Rib-PPaS are depicted in Figures 1 and 2, respectively. Enzymatic synthesis of these analogues was preferred to a possible chemical approach [e.g., Tener & Khorana (1958)] since (1) improved yields would be anticipated, (2) P-Rib-PP synthetase retains catalytic activity with sulfur-substituted substrates, as demonstrated in particular for ribose S-(thiophosphate) (Murray et al, 1969) and ATPaS (Gibson & Switzer, 1980), and (3) the stereospecificity of the S. typhimurium enzyme and the stereochemical course of the reaction were known (Li et al, 1978) (Figure 2A). The latter was an important consideration in designing the synthesis of the P-Rib-PPaS diastereomers and also facilitated their stereochemical assignment (Figure 2B,C).…”

Section: Resultsmentioning

confidence: 99%

“…Aliquots (0.5 mL) were lyophilized, transported to Australia by air, and reconstituted with 50 mM potassium phosphate, pH 7.5. The preparation was rapidly frozen in liquid nitrogen and stored at -70 °C until required 2 Synthesis of the related analogue 5-(thiophospho)ribosyl 1-diphosphate from ribose S-(thiophosphate) and ATP has been described (Murray et al, 1969).…”

Section: Methodsmentioning

confidence: 99%

Phosphorothioate analogs of 5-phosphoribosyl diphosphate: synthesis, purification, and partial characterization

Smithers¹,

O'Sullivan²

1984

Biochemistry

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The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Less than 0.2pmol of radioactive nucleotide/mg of tissue was detected when [8-14C]guanine (50 ,iM) was supplied to chopped cerebrum. The activity of guanine phosphoribosyltransferase assayed by the method of Murray et al (1969), in a crude extract of cerebrum, was 0.25 nmol of GMP formed/min per mg of protein or 16pmol/min per mg of tissue. The inability of guanine to be incorporated into nucleotides is probably due to rapid deamination of the substrate both intracellularly and extracellularly (see below).…”

Section: Purine Ribonucleotide Synthesismentioning

confidence: 99%

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

Wong

Henderson

1972

Biochemical Journal

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The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.

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Analogues of ribose 5-phosphate and 5-phosphoribosyl pyrophosphate. The preparation and properties of ribose 5-phosphorothioate and 5-phosphoribosyl 1-methylenediphosphonate

Cited by 13 publications

References 11 publications

Locations and Functional Roles of Conserved Lysine Residues in Salmonella typhimurium Orotate Phosphoribosyltransferase

Locations and Functional Roles of Conserved Lysine Residues in Salmonella typhimurium Orotate Phosphoribosyltransferase

Phosphorothioate analogs of 5-phosphoribosyl diphosphate: synthesis, purification, and partial characterization

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

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