title: Purification and properties of 4-halobenzoate-coenzyme A ligase from Pseudomonas sp. CBS3.
abstract: The bacterial strain Pseudomonas sp. CBS3 possesses a multi component enzyme system which converts 4-chlorobenzoate to 4-hydroxybenzoate. In the first step 4-chlorobenzoate is activated in a coenzyme A, ATP and Mg(2+)-dependent reaction to 4-chlorobenzoyl-coenzyme A. ATP is cleaved thereby into AMP and pyrophosphate. The involved 4-chlorobenzoate-coenzyme A ligase was purified to apparent homogeneity by a 6-step purification procedure. The native enzyme had an apparent molecular mass of 115000 Da and was composed of two identical polypeptide subunits of 57 kDa. The enzyme displayed an isoelectric point of 5.3. The maximal initial rate of catalysis was achieved in 100mM Tris/HCl or Tricine/NaOH buffer, pH 8.4, at 35 degrees C. Under these conditions the apparent Km values for ATP, coenzyme A and 4-chlorobenzoate were 2.4 to 3.5 mM, 0.11 to 0.19mM and 0.05 to 0.065mM, respectively. Vmax was 111.6 mumol/(min x mg protein). The N-terminal amino-acid sequence was determined. 4-Halobenzoates were preferentially converted to the corresponding thioesters. Therefore, the enzyme was named 4-halobenzoate-coenzyme A ligase.
authors.affiliation: Technische Universität Hamburg-Harburg, Arbeitsbereich Biotechnologie II, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Löffler
authors.forename: R
authors.initials: R
authors.lastname: Müller
authors.forename: F
authors.initials: F
authors.lastname: Lingens
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002723:Chlorobenzoates / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D002723:Chlorobenzoates; D000255:Adenosine Triphosphate; C077846:4-halobenzoate-CoA ligase; D003066:Coenzyme A Ligases; C040768:4-chlorobenzoic acid