title: Self-assembly of Pseudomonas fluorescens lipase into bimolecular aggregates dramatically affects functional properties.
abstract: It has been found that lipase from Pseudomonas fluorescens (PFL) is able to aggregate into bimolecular structures (MW around 66 kD) even at moderate enzyme concentrations. At very low enzyme concentrations and in the presence of detergents, the same enzyme displayed a unimolecular structure with a molecular weight of 33 kD. Both enzyme structures displayed different functional properties. First, the bimolecular structure was much more stable than the unimolecular species (the bimolecular structure maintained over 80% of initial activity after 72 hours at 45 degrees C, while the unimolecular structure retained only around 30% of initial activity after 4 hours of incubation under the same experimental conditions); and the bimolecular form presented a higher optimal T. Second, the unimolecular form showed a much lower K(M) for ethyl butyrate than the bimolecular form. Third, the interfacial activation in biphasic substrate-aqueous milieu was higher for the bimolecular form. Fourth, the unimolecular structure was less active but much more enantioselective than the unimolecular species in the model reaction used. It is proposed that the bimolecular aggregates of PFL might be formed by two open lipase molecules (mutual interfacial activation), in intimate contact, and that the bimolecular form represents an example of "pseudo-quaternary" structure.
authors.affiliation: Department of Biocatalysis, Institute of Catalysis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Fernández-Lorente
authors.forename: José M
authors.initials: JM
authors.lastname: Palomo
authors.forename: Manuel
authors.initials: M
authors.lastname: Fuentes
authors.forename: Cesar
authors.initials: C
authors.lastname: Mateo
authors.forename: José M
authors.initials: JM
authors.lastname: Guisán
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernández-Lafuente
mesh_terms: D004789:Enzyme Activation; D004795:Enzyme Stability; D004952:Esters; D006868:Hydrolysis; D008049:Lipase / Q000096:biosynthesis* / Q000737:chemistry*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010654:Phenylbutyrates / Q000737:chemistry; D011487:Protein Conformation; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000201:enzymology*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D004952:Esters; D046911:Macromolecular Substances; D010654:Phenylbutyrates; C078196:2-hydroxy-4-phenylbutyric acid; D008049:Lipase