Record number :
1450445
Title of article :
The Role of Divalent Magnesium in Activating the Reaction Catalyzed by Orotate Phosphoribosyltransferase
Author/Authors :
Bhatia، نويسنده , , M.B. and Grubmeyer، نويسنده , , C.، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 1993
Pages :
5
From page :
321
To page :
325
Abstract :
Orotate phosphoribosyltranferase (OPRTase) catalyzes the formation of orotidine 5′-monophosphate from the nitrogenous base orotate and α-D-5-phospboribosyl-1-pyrophosphate (PRPP). While it is known that Mg2+ is necessary for catalysis, the mechanism of activation of the phosphoribosyl transfer by Mg2+ remains unclear. The divalent cation may activate the phosphoribosyl transfer by binding to either or both substrates PRPP and orotate or/and the enzyme. In this work we chose to explore the role of divalent magnesium in activating the phosphoribosyl transfer in bacterial OPRTase. Studies on the effect of Mg2+ on the OPRTase-catalyzed reaction indicated that the divalent metal was necessary for catalysis. A maximal rate of 70 units/mg was achieved at 2 mM MgCl2. Mn2+ could replace Mg2+ as the divalent metal. Orotate methyl ester (OAME) and uracil, neither of which form chelates with divalent metal, were found to be substrates for OPRTase. The KM for OAME and uracil were 190 μM and 2.63 mM and kcat/KM were 0.91 × 105 and 6 M−1 s−1, respectively. These values compare with a KM of 27 μM for orotate, 44 μM for PRPP, and a kcat/KM of 1.3 × 106 M−1 s−1 for orotate. Spectroscopic studies failed to reveal the existence of Mg2+-orotate complexes. Thus we have concluded that an orotate-metal complex is not necessary for OPRTase catalysis. Metal-enzyme binding studies indicate that only weak metal-enzyme complexes may form in bacterial OPRTase. Thus the role of divalent metal in bacterial OPRTase must be to bind PRPP.
Journal title :
Archives of Biochemistry and Biophysics
Serial Year :
1993
Link To Document :
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