Abstract
The role of Mg ions in the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction have been studied using accurate values of proton and Mg stability constants of phosphoribosylpyrophosphate (P-Rib-PP) determined from pH titration data. The results obtained favor the conclusion that the dimagnesium salt of P-Rib-PP is the true substrate of the enzyme. The other species of P-Rib-PP do not appreciably affect the initial reaction rate. The inhibition of the hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction observed at high MgCl2 concentration can be attributed to a competitive inhibition of Mg2+ with respect to the dimagnesium salt of P-Rib-PP, suggesting that these ionic species bind to the same enzyme form. At a fixed [P-Rib-PPtot], the concentration of its dimagnesium complex is a sigmoidal function of MgCl2 concentration, suggesting that caution must be employed in the interpretation of sigmoidal saturation curves for P-Rib-PP-utilizing enzymes when low and not constant concentrations of the divalent cation are used.
Highlights
From the InstituteofBiologica1 Chemistry and Instituteof Rheumatology, University of Rome and Center of Molecular Biology, Consiglio Nationale delle Ricerche, Rome, Italy
The role of M g ionsinthehypoxanthineguanine curve for hypoxanthine guanine phosphoribosyltransferase phosphoribosyltransferase-catalyzed reactiohnave has been attributedeitherto allosteric effects (13) or to been studied using accurate values of proton and M g competition of one ionic species of P-Rib-PP with the true stabilityconstants of phosphoribosylpyrophosphate substrate of the enzyme (11).varying magnesium (P-Rib-PP) determined frompH titration data.The re- ion concentration differences in the initial velocity and inhisults obtained favor the conclusion that the dimagne- bition patterns have been obtained studying steady state sium salt of P-Rib-PP is the true substrate of the en- kinetics of hypoxanthine guanine phosphoribosyltransferase zyme
The inhibition of the ferase kinetics in the presence of a large excess of magnesium hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction observed at highMgClz concentration can be attributed to a competitive inhibition of M&' with respectto the dimagnesium saltof P-Rib-PP, suggestingthat these ionic specbiiensd to the same enzyme form
Summary
From the InstituteofBiologica Chemistry and Instituteof Rheumatology, University of Rome and Center of Molecular Biology, Consiglio Nationale delle Ricerche, Rome, Italy. The inhibition of the ferase kinetics in the presence of a large excess of magnesium hypoxanthine guanine phosphoribosyltransferase-catalyzed reaction observed at highMgClz concentration can be attributed to a competitive inhibition of M&' with respectto the dimagnesium saltof P-Rib-PP, suggestingthat these ionic specbiiensd to the same enzyme form. In the case of P-Rib-PP amidotransferase, the substratevelocity plot with respect to P-RibP P appeared sigmoidal at low magnesium ion concentrations using the enzyme obtained from human placenta (2) and EXPERIMENTAL PROCEDURES. In the case of hypoxanthine guanine phosphoribosyltransferase, the plot of initial velocity uersus P-Rib-PP concentration appeared hychromatography according to Wood (18) showed no significant impurity in the commercial sample of P-Rib-PP after staining with ammonium molybdate (18).All other reagents were high purity commercial samples from Merck A.G., Fluka A.G., and Boehringer A.G. All experiments were carried out a t 25°C in the presence of 0.17 perbolic at high magnesium ion concentration and sigmoidal M NaC1. Varying triethanolamine concentration from to 7 X IO-' M in the presence of 5 X M MgCL and 3.4 X M P-Rib-PP,no appreciable variation
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