Studies of the binding of the magnesium-phosphoribosyl 1-pyrophosphate complex to orotate phosphoribosyltransferase from yeast
Both C/sup 14/- and /sup 13/C-phosphoribosyl 1-pyrophosphate molecules, labeled at the C'-1 position, were synthesized biochemically with bacterial ribokinase and PRibPP synthetase activities. A flow dialysis experiment characterized the dissociation constant (33 ..mu..M) for the OPRTase-C/sup 14/PRibPP-Mg(II) complexation and defined 2 PRibPP binding sites per enzyme dimer. Moreover, labeled-PRibPP was observed to elute with a 50 ..mu..g sample of OPRTase through Sephadex Superfine G-25, suggesting that one form of the label is bound more tightly to the enzyme then the value of Kd would suggest. Samples of /sup 13/C-PRibPP, were characterized with /sup 13/C-NMR. A doublet appeared in the proton-decoupled PRibPP spectrum. The addition of a saturating level of Mg(II) to PRibPP resulted in a 0.1 ppm upfield shift. The addition of 0.6 mM OPRTase to a sample containing 0.9 mM /sup 13/C-PRibPP and 4 mM Mg (II) resulted in a disappearance of the doublet. Upon further addition of 1 mM /sup 13/C-PRibPP, four resonances were observed and tentatively assigned as follows: beta-/sup 13/C ribose 5' phosphate OPRTase bound-/sup 13/C-PRibPP, alpha-/sup 13/C-ribose 5' phosphate and a /sup 13/C-phosphoribosyl-OPRTase adduct. These assignments are consistent with the OPRTase ping pong kinetic mechanism, during which the pyrophosphate portion of PRibPP dissociates from the active site prior to orotate binding, and during which PRibPP undergoes hydrolysis within the time frame of the NMR experiment.
- Research Organization:
- City College of CUNY, NY
- OSTI ID:
- 6074935
- Report Number(s):
- CONF-870644-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 46:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CARBON 13
CARBON 14 COMPOUNDS
CARBON ISOTOPES
CHEMICAL PREPARATION
CHEMICAL SHIFT
ENZYME ACTIVITY
ENZYMES
EVEN-ODD NUCLEI
FUNGI
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEI
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PLANTS
PROTEINS
PYROPHOSPHATES
REACTION KINETICS
RECEPTORS
SPECTRA
STABLE ISOTOPES
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
YEASTS