Chemical characterization of phosphoribosylamine, a substrate for newly discovered trifunctional protein containing glycineamide ribonucleotide synthetase activity.
PRA has been characterized for the first time using 13C-NMR spectroscopy. Incubation of [1-13C]ribose-5-phosphate with NH3 results in the production of 38:62 alpha:beta anomeric mixture of PRA, alpha,beta ribose-5-phosphate and variable amounts of dimeric materials. NMR studies at various pHs allowed determination of the pH independent Kequi = 0.95 +/- 0.14 M-1 for this reaction. In addition, using magnetization transfer NMR methodology the rate of conversion of alpha to beta PRA was determined to be 44 sec-1 at 37 degrees C (pH 8.0). The rates of formation (from ribose-5-phosphate and NH3) and degradation of PRA were also measured using E. coli GAR synthetase (recently cloned, overproduced and purified to homogeneity) as a trap. Determination of these rates allowed an independent and accurate measurement of Kequi = 2.7 M-1. In addition, in close agreement with early studies of Nierlich and Magasanik, the half life of PRA at 37 degrees C and pH 7.5 was determined to be 35 sec. Characterization of the chemical stability of PRA and Kequi for ribose-5-phosphate, NH3 with PRA will now allow detailed kinetic analysis of the newly discovered trifunctional protein containing GAR synthetase activity in addition to AIR synthetase and GAR transformylase activities. Comparison of the properties of the 110 kd GAR synthetase and an independently isolated 54 kd GAR synthetase are reported. Experiments are underway to investigate the possibility that unstable intermediates such as PRA are not released into solution, but that the transfer is mediated by specific protein-protein interactions between GAR synthetase and PRPP amidotransferase.
Cheng
Y S
YS
University of Wisconsin-Madison 53706.
Murray
M
M
Schendel
F
F
Otvos
J
J
Wehrli
S
S
Stubbe
J
J
D000641:Ammonia; D019731:Carbon-Nitrogen Ligases; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008025:Ligases / Q000302:isolation & purification*; D010428:Pentosephosphates; D012267:Ribosemonophosphates
D010428:Pentosephosphates; D012267:Ribosemonophosphates; C008891:phosphoribosylamine; C031626:ribose-5-phosphate; D000641:Ammonia; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022380:phosphoribosylaminoimidazole synthase; C022387:phosphoribosylamine-glycine ligase