Purification and characterization of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.
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 Km for adenine and PRPP were 6 microM and 15 microM, respectively and the Vmax was 15 micromol/min. These kinetic constants are comparable to the constants of APRT from other organisms.
Alfonzo
J D
JD
Howard Hughes Medical Institute, MacDonald Research Laboratories, University of California at Los Angeles, 90024, USA.
Sahota
A
A
Taylor
M W
MW
D000225:Adenine / Q000378:metabolism / Q000494:pharmacology; D000228:Adenine Phosphoribosyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D002412:Cations / Q000494:pharmacology; D002845:Chromatography; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D017421:Sequence Analysis; D013696:Temperature
D002412:Cations; D004791:Enzyme Inhibitors; D000249:Adenosine Monophosphate; D010754:Phosphoribosyl Pyrophosphate; D000228:Adenine Phosphoribosyltransferase; D000225:Adenine