Purification and some properties of delta 2-isopentenylpyrophosphate:5'AMP delta 2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum.
delta 2-Isopentenylpyrophosphate:5'AMP delta 2-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from delta 2-isopentenylpyrophosphate and 5'AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5'AMPox-red AH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 +/- 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn2+, Zn2+, and Mg2+ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5'AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrates, being 60-80% as effective as 5'AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mM Zn2+, and 25 degrees C) the Km values for 5'AMP and delta 2-isopentenylpyrophosphate were 1.0 X 10(-7)M and 2.2 X 10(-6)M, respectively.
D019883:Alkyl and Aryl Transferases; D002845:Chromatography / Q000379:methods; D004023:Dictyostelium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D014166:Transferases / Q000302:isolation & purification*
D008670:Metals; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase