title: A new enzyme model for enantioselective esterases based on molecularly imprinted polymers.
abstract: An efficient enzyme model exhibiting enantioselective esterase activity was prepared by using molecular imprinting techniques. The enantiomerically pure phosphonic monoesters 4 L and 5 L were synthesized as stable transition-state analogues. They were used as templates connected by stoichiometric noncovalent interactions to two equivalents of the amidinium binding site monomer 1. After polymerization and removal of the template, the polymers were efficient catalysts for the hydrolysis of certain nonactivated amino acid phenylesters (2 L, 2 D, 3 L, 3 D) depending on the template used. Imprinted catalyst IP4 (imprinted with 4 L) enhanced the hydrolysis of the corresponding substrate 2 L by a factor of 325 relative to that of a buffered solution. Relative to a control polymer containing the same functionalities, prepared without template 4 L, the enhancement was still about 80-fold, showing the highest imprinting effect up to now. In cross-selectivity experiments a strong substrate selectivity of higher than three was found despite small differences in the structure of the substrate and template. Plots of initial velocities of the hydrolysis versus substrate concentration showed typical Michaelis-Menten kinetics with saturation behavior. From these curves, the Michaelis constant K(M) and the catalytic constant k(cat) can be calculated. The enantioselectivity shown in these values is most interesting. The ratio of the catalytic efficiency k(cat)/K(M), between the hydrolysis of 2 L- and 2 D-substrate with IP4, is 1.65. This enantioselectivity derives from both selective binding of the substrate (K(M)L/K(M)D=0.82), and from selective formation of the transition state (k(cat)L/k(cat)D=1.36). Thus, these catalysts give good catalysis as well as high imprinting and substrate selectivity. Strong competitive inhibition is caused by the template used in imprinting. This behavior is also quite similar to the behavior of natural enzymes, for which these catalysts are good models.
authors.affiliation: Institute of Organic Chemistry and Macromolecular Chemistry Heinrich-Heine-University Düsseldorf, Universitätsstrasse 1 40225 Düsseldorf, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Emgenbroich
authors.forename: Günter
authors.initials: G
authors.lastname: Wulff
mesh_terms: D002021:Buffers; D002384:Catalysis; D004950:Esterases / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D008968:Molecular Conformation; D015394:Molecular Structure; D011108:Polymers / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002021:Buffers; D011108:Polymers; D004950:Esterases; D007930:Leucine; D014633:Valine