Adenosine 5'-tetraphosphate phosphohydrolase from yellow lupin seeds: purification to homogeneity and some properties

• Published in: Biochemical journal 1997-11, Vol.328 ( Pt 1) (1), p.257-262
• Main Authors: Guranowski, A, Starzyńska, E, Brown, P, Blackburn, G M
• Format: Article
• Language: English
• Subjects: Acid Anhydride Hydrolases - antagonists & inhibitors; Acid Anhydride Hydrolases - chemistry; Acid Anhydride Hydrolases - isolation & purification; Acid Anhydride Hydrolases - metabolism; Adenine Nucleotides - metabolism; Calcium - pharmacology; Cations, Divalent; Fluorides - pharmacology; Hydrogen-Ion Concentration; Kinetics; Plant Proteins - antagonists & inhibitors; Plant Proteins - chemistry; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Protein Conformation; Substrate Specificity; Sulfhydryl Compounds - pharmacology; Zinc - pharmacology
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3280257

Adenosine 5'-tetraphosphate phosphohydrolase (EC 3.6.1.14) has been purified to homogeneity from the meal of yellow lupin (Lupinus luteus) seeds. The enzyme is a single polypeptide chain of 25+/-1 kDa. It catalyses the hydrolysis of a nucleoside 5'-tetraphosphate to a nucleoside triphosphate and orthophosphate, and hydrolysis of tripolyphosphate but neither pyrophosphate nor tetraphosphate. A divalent cation, Mg2+, Co2+, Ni2+ or Mn2+, is required for these reactions. The pH optimum for hydrolysis of adenosine 5'-tetraphosphate (p4A) is 8.2, Vmax is 21+/-1.7 micromol/min per mg of protein and the Km for p4A is 3+/-0.6 microM. At saturating p4A concentrations, the rate constant for the reaction is 8.5+/-0.7 s-1 [at 30 degrees C, in 50 mM Hepes/KOH (pH8.2)/5 mM MgCl2/0.1 mM dithiothreitol]. p4A and guanosine 5'-tetraphosphate are hydrolysed at the same rate. Adenosine 5'-pentaphosphate (p5A) is degraded 1/200 as fast and is converted into ATP and two molecules of orthophosphate, which are liberated sequentially. This contrasts with the cleavage of p5A by the lupin diadenosine tetraphosphate hydrolase (EC 3.6.1.17), which gives ATP and pyrophosphate. Zn2+, F- and Ca2+ ions inhibit the hydrolysis of p4A with I50 values of 0.1, 0.12 and 0.2 mM respectively.