Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: kinetic analysis with chromium (III) pyrophosphate.
The reactions catalyzed by orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) from yeast differ in the kinetic mechanisms by which they are activated by divalent metal ions. Moreover, whereas OPRTase is activated specifically by Mg(II) or Mn(II), the reactions catalyzed by HGPRTase can utilize a wider range of divalent metal ions, including Mg(II), Mn(II), Co(II), and Zn(II). In this report we describe the results of a kinetic analysis of the effects of the addition of Cr(III) pyrophosphate (Cr-PPi) to the OPRTase and HGPRTase assay solutions, which delineates further the differences between these enzyme activations by metal ions. (1) Cr-PPi is an effective competitive inhibitor of the OPRTase catalysis, when the steady-state forward velocity of orotidine monophosphate (OMP) formation is examined over a range of phosphoribosyl alpha-pyrophosphate (PRibPP) concentrations, whereas pyrophosphate (PPi) has been reaffirmed to be a noncompetitive product inhibitor under the same conditions. (2) Cr-PPi itself serves as a substrate for the OPRTase-catalyzed reverse pyrophosphorolysis of OMP and does not inhibit the utilization of PPi as substrate during this reaction. (3) In contrast, Cr-PPi, at concentrations as high as 6 mM, has no effect on the HGPRTase-catalyzed formation of inosine monophosphate, whereas the inhibition exhibited by PPi during this reaction is noncompetitive but defined by two sets of lines in the double reciprocal plot of the initial velocity versus 1/PRibPP. (4) Cr-PPi is not a substrate for the HGPRTase-catalyzed pyrophosphorolysis of IMP under the conditions of these assay procedures.
Syed
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Department of Chemistry, City College, City University of New York, New York 10031.
Sloan
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D055598:Chemical Phenomena; D002621:Chemistry; D002857:Chromium / Q000494:pharmacology*; D011756:Diphosphates / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism*; D007700:Kinetics; D009962:Orotate Phosphoribosyltransferase / Q000378:metabolism*; D010430:Pentosyltransferases / Q000378:metabolism*; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D011756:Diphosphates; D002857:Chromium; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase