Orotate phosphoribosyltransferase from yeast: studies of the structure of the pyrimidine substrate binding site.
The pH dependencies of both the forward and reverse orotate phosphoribosyltransferase (ORPTase)-catalyzed reactions have been examined and determined to be dissimilar, with maximal activity for the forward reaction near to pH 8. The maximal activity of the reverse pyrophosphorolysis was observed between pH 6.5 and 7.5. Appropriate pK values were determined using computer fitting exercises. One such pK value (equal to 8.6) suggested the presence of lysine residues at the OPRTase active site. Incubations of OPRTase with the substrate analog, uracil 6-aldehyde, in the presence of sodium borohydride, suggested that this compound is a covalent modifier of OPRTase lysine residues, and substrate protection studies provided evidence that the affected lysine residues were located near to both the phosphoribosyl 1-pyrophosphate (PRibPP) and the orotate binding sites. Similar studies with pyridoxal 5-phosphate and labeled sodium borohydride as modifiers have revealed that two modified active site lysine residues per OPRTase subunit account for the loss of 90% of the enzymatic activity with this reagent. We suggest that essential lysine residues, along with divalent metal ions, are located at the OPRTase active site, and form ion-pair bonds with anionic PRibPP and orotate as these substrates bind to the enzyme. We also report that 5-azaorotate is an alternate substrate for OPRTase (Km = 75.5 +/- 0.1 microM) leading to formation of an unstable nucleotide product).
Ashton
R W
RW
Department of Chemistry, City College of the City University of New York, New York 10031.
Strauss
R S
RS
Chung
S H
SH
Sloan
D L
DL
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009962:Orotate Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000345:Affinity Labels; D000596:Amino Acids; D008345:Manganese; D011732:Pyridoxal Phosphate; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D008274:Magnesium