ATP:AMP phosphotransferase from baker's yeast. Purification and properties.
An improved homogeneous preparation of adenylate kinase (ATP:AMP phosphotransferase, ATP + AMP in equilibrium 2 ADP) from baker's yeast was attained by extraction using ethyl acetate and successive column chromatography on Affi-Gel blue, Sephadex G-100, phosphocellulose and Sephacryl S-200. The overall purification is about 670-fold with a yield of 23% and final specific activity of 1900 units/mg protein. The enzyme preparation is a single band in isoelectrofocusing with a pI of 5.7. By sodium dodecyl sulfate gel electrophoresis and gel chromatography the molecular weight is 27 500. Among the nucleoside monophosphates investigated (AMP, CMP, GMP, IMP and UMP) only AMP reacts with ATP (dATP). ATP (dATP) is about one order of magnitude more active than CTP, GTP, ITP and UTP. The enzyme catalyzes only in the presence of a divalent metal cation, namely Mg2+, Ca2+, Co2+, Mn2+ and Ni2+, and the reaction rate is maximal at about 0.5 M NaCl. The binding of the substrates also takes place in the absence of metal. The dissociation constants for ATP, MgATP, CTP, GTP, UTP and AMP are 3.4, 4.2, 18.5, 17.2, 23.8 and 39.3 microM respectively. The amino acid composition of the purified enzyme is: 32 aspartic acid + asparagine, 12 threonine, 12 serine, 27 glutamic acid + glutamine, 16 proline, 21 glycine, 24 alanine, 11 valine, 9 methionine, 17 isoleucine, 25 leucine, 4 tyrosine, 7 phenylalanine, 2 half-cystine (no free sulfhydryl), 23 lysine, 6 histidine, 10 arginine and 2 tryptophan, totalling 260 residues.
D000263:Adenylate Kinase / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D000596:Amino Acids; D002413:Cations, Divalent; D010770:Phosphotransferases; D000263:Adenylate Kinase