Molecular recognition of an acyl-enzyme intermediate on the lipase B of Candida antarctica

SEBASTIÁN E. COLLINS; MARÍA V. TOLEDO; LAURA E. BRIAND; CARLOS R. LLERENA-SUSTER

Encuentro; 252nd American Chemical Society Meeting; 2016

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are hydrolytic enzymes whose biological function in bacteria, fungi, plants and higher animals, is to catalyze the hydrolysis of triglycerides to obtain free fatty acids and glycerol. These enzymes are of great utility in various reactions and numerous industrial processes due to their ability to catalyze the reverse reactions of esterification, transesterification and interesterification. Pharmaceutical industry applies the enantioselectivity property of lipases for preparing different pharmaceuticals and fine chemicals containing a chiral center. Among the racemic substances, 2-arylpropionic acids derivatives are an important group of non-steroidal antiinflamatory pharmaceuticals (NSAIDs) used to treat pain and inflammation associated with tissue injury. While they are commercially available as racemic mixtures, their pharmacological activity remains mainly on the S(+)-enantiomer; the R(-)-isomer may also present unwanted physiological side effects and toxicity.In the last 20 years lipases have been widely applied in kinetic resolution of this racemic mixtures with the aim of obtaining optically pure S(+)-2-arylpropionic acids. Lipase B from Candida antarctica (CALB) has been one of the most used. CALB is composed of 317 amino acids and a molecular weight of 33 kDa. The active site is a catalytic triad composed by Serine (S) 105, Aspartic Acid (D) 187 and Histidine (H) 224, and an oxyanion hole formed by Threonine (T) 40 and Glutamine (Q) 106 are found. The mechanism of action of this enzyme has been described as a Ping Pong Bi Bi with the formation of two tetrahedral intermediates and an acyl-enzyme complex.In the present investigation, molecular species involved in the acyl-enzyme complex between the R/S-ketoprofen with the lipase B from Candida antarctica are studied by in situ Time-Resolved ATR-FTIR.