ATP Sulfurylase from Higher Plants: Kinetic and Structural Characterization of the Chloroplast and Cytosol Enzymes from Spinach Leaf

• Published in: Archives of biochemistry and biophysics Vol. 307; no. 2; pp. 272 - 285
• Main Authors: Renosto, F., Patel, H.C., Martin, R.L., Thomassian, C., Zimmerman, G., Segel, I.H.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.12.1993; Elsevier
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Adenosine Phosphosulfate; Adenosine Phosphosulfate - metabolism; ADENOSINE TRIPHOSPHATE; Adenosine Triphosphate - metabolism; ADENOSINTRIFOSFATO; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; antagonists & inhibitors; Biological and medical sciences; CHLOROPLASTE; chloroplasts; Chloroplasts - enzymology; CITOPLASMA; CLOROPLASTO; CYTOPLASME; cytosol; Cytosol - enzymology; Diphosphates; Diphosphates - metabolism; enzyme activity; Enzymes and enzyme inhibitors; enzymology; FEUILLE; Fluorides; Fluorides - metabolism; Fundamental and applied biological sciences. Psychology; HOJAS; Hydrogen-Ion Concentration; Isoenzymes; Isoenzymes - isolation & purification; isolation & purification; Kinetics; leaves; Ligands; metabolism; Models, Chemical; Molecular Sequence Data; Molybdenum; Molybdenum - metabolism; Nucleotides; Nucleotides - pharmacology; Peptide Mapping; pharmacology; Phosphates; Phosphates - metabolism; PURIFICACION; PURIFICATION; Sequence Analysis; SPINACIA OLERACEA; Substrate Specificity; SULFATE; Sulfate Adenylyltransferase; Sulfate Adenylyltransferase - antagonists & inhibitors; Sulfate Adenylyltransferase - isolation & purification; Sulfate Adenylyltransferase - metabolism; sulfates; Sulfates - metabolism; SULFATOS; TRANSFERASAS; TRANSFERASE; Transferases; Vegetables; Vegetables - enzymology; Enzyme; Isozyme; Transferases; Spinacia oleracea; Subunit; Chenopodiaceae; Nucleotidyltransferases; Sulfate adenyltransferase; Enzymatic activity; Dicotyledones; Angiospermae; Spermatophyta; Kinetic parameter; Chloroplast; Comparative study; Structural analysis
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1993.1590

Two forms of ATP sulfurylase were purified from spinach leaf. The major (chloroplast) form accounts for 85 to 90% of the total leaf activity (0.03 ± 0.01 adenosine-5′-phosphosulfate (APS) synthesis units × gram fresh weight −1). Both enzyme forms appear to be tetramers composed of 49- to 50-kDa subunits with the minor (cytosolic) form being slightly larger than the chloroplast form. The specific activities (units × milligram protein −1) of the chloroplast form at pH 8.0, 30°C, were as follows:APS synthesis, 16; molybdolysis, 229; ATP synthesis, 267; selenolysis, 4.1; fluorophosphate activation, 11. Kinetic constants for the physiological reaction were as follows: K mA = 0.046 mM, K ia = 0.85 mM, K mB = 0.25 mM, K mQ = 0.37 μM, K iq = 64-85 nM, and K mP = 10 μM, where A = MgATP, B = SO 2− 4, P = total PP i at 5 mM Mg 2+, and Q = APS. The kinetic constants for molybdolysis were similar to those of the APS synthesis reaction. The kinetic constants of the minor (cytosol) form were similar to those of the major form with two exceptions: (a) The molybdolysis activity was 120 units × milligram protein −1, yielding a V max (ATP synthesis)/ V max (molybdolysis) ratio close to 2 (compared to about uaity for the chloroplast form) and (b) K mA was greater (0.24 and 0.15 mM for APS synthesis and molybdolysis, respectively). Initial velocity measurements (made over an extended range of MgATP and SO 2− 4 concentrations), product inhibition studies (by initial velocity methods and by reaction progress curve analyses), dead end inhibition studies (with monovalent and divalent oxyanions), and k cat/ K m comparisons (for SO 2− 4 and MoO 2− 4) support a random AB-ordered PQ kinetic mechanism in which MgATP and SO 2− 4 bind in a highly synergistic manner. Equilibrium binding studies indicated the presence of one APS site per subunit. HPLC elution profiles of chymotryptic and tryptic peptides were essentially the same for both enzyme forms. The N-terminal sequence of residues 5-20 of the cytosol enzyme was identical to residues 1-16 of the chloroplast enzyme.