A stereochemical investigation of the cyclisation ofD-glucose-6[(R)-¹⁶O,¹⁷O,¹⁸O]phosphate and adenosine-5′[(R)-¹⁶O,¹⁷O,¹⁸O]phosphate

The stereochemical course of phosphoryl transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected Escherichia coli B)

“…The chirality at the phosphorus atom was analysed by 3p n.m.r. spectroscopy after cyclization (which occurs with inversion of configuration) and esterification as described previously (Jarvest et al, 1980(Jarvest et al, , 1981Cullis et al, 198 la); the 31P n.m.r. spectrum is shown in Fig.…”

“…1) n.m.r. spectroscopy after cyclization and esterification as previously described (Jarvest et al, 1980(Jarvest et al, , 1981Cullis et al, 198 la). 3p n.m.r.…”

“…The sequential mechanism is certainly consistent with the lack of polynucleotide kinase-catalysed ADP-ATP exchange, but substrate synergism (Bridger et al, 1968) We have developed a method based on 31P n.m.r. spectroscopy for analysing the chirality at the phosphorus atom of [160,170,1801ATP (Jarvest et al, 1980(Jarvest et al, , 1981Cullis et al, 1981a). If 3'-AMP is used as the co-substrate and the 3'-phosphate subsequently removed with nuclease P1, analysis of the chirality at the phosphorus atom of the residual 5'-[160,170,180ATP should allow the stereochemical course of the phosphoryl transfer catalysed by polynucleotide kinase to be determined.…”

A Stereochemical and Positional Isotope‐Exchange Study of the Mechanism of Activation of Methionine by Methionyl‐tRNA Synthetase from Escherichia coli

“…Inorganic pyrophosphatase was also present in the reaction mixture to hydrolyse the magnesium pyrophosphate generated and so assist the overall reaction (Scheme 1). The [l60, 170, "OIAMP was isolated from the reaction mixture and its chirality at phosphorus determined by our established procedure after cyclization and methylation [23]. The 31P NMR spectrum is shown in Fig.…”

“…The reaction was monitored by assaying for residual ATP by the coupled reactions of hexokinase and glucose-6-phosphate dehydrogenase [22]. When the reaction was complete (8 h), the solution was applied to a column (1.5 cm x 30 cm) of DEAESephadex A25, which had been equilibrated with triethylammonium bicarbonate buffer (50 mM, pH 7.6) and eluted with a linear gradient of triethylammonium bicarbonate buffer (50-200 mM, pH 7.6, 2.5 I).-The [l60, 1 7 0 , "OIAMP (34 pmol) was freed from buffer by addition and evaporation of methanol (3 x 10 ml) and then cyclized and methylated for analysis by 31P NMR spectroscopy of the chiralty at phosphorus [23].…”