A monolayer and bulk study on the kinetic behavior of Pseudomonas glumae lipase using synthetic pseudoglycerides.
A heat-stable lipase from Pseudomonas glumae was purified to homogeneity. Its positional and stereospecific properties were investigated and compared with those of the well-known porcine pancreatic lipase. The kinetic properties of both enzymes were determined by use of six isomeric synthetic pseudoglycerides all composed of a single hydrolyzable fatty acyl ester bond and two lipase-resistant groups: one acylamino and one ether function. Two enzyme assay techniques were applied: a detergent-free system, the monomolecular surface film technique, and the pH-stat technique using clear micellar solutions of substrate in the presence of Triton X-100. Regarding the cleavage of primary ester bonds, P. glumae lipase possesses no stereopreference. In contrast, a large stereopreference in favor of the R-isomer is found for the hydrolysis of secondary ester bonds. Secondary ester bonds are efficiently cleaved by the lipase, which makes it of potential interest for enzymatic synthetic purposes. For the hydrolysis of this R-isomer a correlation between the experimental catalytic turnover rate and the binding constant for micelles was observed. The kinetic data of P. glumae lipase have been analyzed in terms of the scooting and hopping models for the action of lipolytic enzymes [Upreti, G.C., & Jain, M.K. (1980) J. Membr. Biol. 55, 113-121]. The results presented in this study are best explained by assuming that glumae lipase leaves the interface after a limited number of catalytic cycles.
Deveer
A M
AM
Department for Enzymology and Protein Engineering, CBLE, Utrecht, The Netherlands.
Dijkman
R
R
Leuveling-Tjeenk
M
M
van den Berg
L
L
Ransac
S
S
Batenburg
M
M
Egmond
M
M
Verheij
H M
HM
de Haas
G H
GH
D000818:Animals; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D008970:Molecular Weight; D017830:Octoxynol; D010179:Pancreas / Q000201:enzymology; D011092:Polyethylene Glycols; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology*; D013237:Stereoisomerism; D013552:Swine; D014280:Triglycerides / Q000138:chemical synthesis / Q000494:pharmacology*
D014280:Triglycerides; D011092:Polyethylene Glycols; D017830:Octoxynol; D008049:Lipase