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The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase.
Arch Biochem Biophys. 1993 Jun; 303(2):321-5.AB

Abstract

Orotate phosphoribosyltranferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate from the nitrogenous base orotate and alpha-D-5-phosphoribosyl-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 microM and 2.63 mM and kcat/KM were 0.91 x 10(5) and 6 M-1 s-1, respectively. These values compare with a KM of 27 microM for orotate, 44 microM for PRPP, and a kcat/KM of 1.3 x 10(6) M-1 s-1 for orotate. Spectroscopic studies failed to reveal the existence of Mg(2+)-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.

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Publisher Full Text

Authors+Show Affiliations

Bhatia MB
Department of Biology, New York University, New York 10003.
Grubmeyer C
No affiliation info available

MeSH

Cations, DivalentEnzyme ActivationMagnesiumManganeseOrotate PhosphoribosyltransferaseOrotic AcidPhosphoribosyl PyrophosphateSalmonella typhimuriumSubstrate SpecificityUracil

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

7685580

Citation

Bhatia, M B., and C Grubmeyer. "The Role of Divalent Magnesium in Activating the Reaction Catalyzed By Orotate Phosphoribosyltransferase." Archives of Biochemistry and Biophysics, vol. 303, no. 2, 1993, pp. 321-5.
Bhatia MB, Grubmeyer C. The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase. Arch Biochem Biophys. 1993;303(2):321-5.
Bhatia, M. B., & Grubmeyer, C. (1993). The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase. Archives of Biochemistry and Biophysics, 303(2), 321-5.
Bhatia MB, Grubmeyer C. The Role of Divalent Magnesium in Activating the Reaction Catalyzed By Orotate Phosphoribosyltransferase. Arch Biochem Biophys. 1993;303(2):321-5. PubMed PMID: 7685580.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase. AU - Bhatia,M B, AU - Grubmeyer,C, PY - 1993/6/1/pubmed PY - 1993/6/1/medline PY - 1993/6/1/entrez SP - 321 EP - 5 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 303 IS - 2 N2 - Orotate phosphoribosyltranferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate from the nitrogenous base orotate and alpha-D-5-phosphoribosyl-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 microM and 2.63 mM and kcat/KM were 0.91 x 10(5) and 6 M-1 s-1, respectively. These values compare with a KM of 27 microM for orotate, 44 microM for PRPP, and a kcat/KM of 1.3 x 10(6) M-1 s-1 for orotate. Spectroscopic studies failed to reveal the existence of Mg(2+)-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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/7685580/The_role_of_divalent_magnesium_in_activating_the_reaction_catalyzed_by_orotate_phosphoribosyltransferase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(83)71290-7 DB - PRIME DP - Unbound Medicine ER -