Purification and characterization of adenine phosphoribosyltransferase from Saccharomyces cerevisiae

Juan D. Alfonzo, Amrik Sahota, Milton W. Taylor

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Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold. The enzyme catalyzes the Mg-dependent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP) to yield AMP. The purification procedure included anion exchange chromatography, chromatofocusing and gel filtration. Elution of the enzyme from the chromatofocusing column indicated a pI value of 4.7. The molecular mass for the native enzyme was 50 kDa; however, upon electrophoresis under denaturing conditions two bands of apparent molecular mass of 29 and 20 kDa were observed. We have previously reported the presence of two separate coding sequences for APRT, APT1 and APT2 in S. cerevisiae. The appearance of two bands under denaturing conditions suggests that, unlike other APRTs, this enzyme could form heterodimers. This may be the basis for substrate specificity differences between this enzyme and other APRTs. Substrate kinetics and product inhibition patterns are consistent with a ping-pong mechanism. The K(m) for adenine and PRPP were 6 μM and 15 μM, respectively and the V(max) was 15 μmol/min. These kinetic constants are comparable to the constants of APRT from other organisms.

Original languageEnglish (US)
Pages (from-to)173-182
Number of pages10
JournalBiochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
Issue number2
StatePublished - Sep 5 1997
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology


  • Adenine phosphoribosyltransferase
  • K(m)
  • Kinetics
  • Purification
  • Purine metabolism
  • V(max)
  • Yeast

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