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Báo cáo khoa học: Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263

Kinetic experiments with a substrate series of phenylacetyl-arylamides reveal that at least one polar group in the amine moiety is required for the proper orientation of the substrate in the large nucleophile-binding subsite of penicillin acylase ofEscherichia coli. Quantum mechanical molecular model-ling of enzyme–substrate interactions in the enzyme active site shows that in the case of substrates lacking local sym-metry, the productive binding implies two nonsymmetrical arrangements with respect to the two positively charged guanidinium residues of ArgA145 and ArgB263 | Eur. J. Biochem. 271 2272-2279 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04170.x Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263 Maya Guncheva1 Ivaylo Ivanov2 Boris Galunsky3 Nicolina Stambolieva1 and Jose Kaneti1 institute of Organic Chemistry with Centre of Phytochemistry Bulgarian Academy of Sciences Sofia Bulgaria laboratory of Bioorganic Synthesis Faculty of Biology University of Sofia Bulgaria 3Department of Biotechnology II Technical University Hamburg-Harburg Germany Kinetic experiments with a substrate series of phenylacetylarylamides reveal that at least one polar group in the amine moiety is required for the proper orientation of the substrate in the large nucleophile-binding subsite of penicillin acylase of Escherichia coli. Quantum mechanical molecular modelling of enzyme-substrate interactions in the enzyme active site shows that in the case of substrates lacking local symmetry the productive binding implies two nonsymmetrical arrangements with respect to the two positively charged guanidinium residues of ArgA145 and ArgB263. This indicates a crucial role of the specified arginine pair in the substrate- and stereoselectivity of penicillin acylase. Keywords enzyme kinetics molecular modelling nucleophile specificity and stereoselectivity penicillin acylase. Penicillin G acylases PA EC 3.5.1.11 from different sources have been widely studied because of their application as industrial biocatalysts for hydrolytic and synthetic transformations in the production of semisynthetic b-lactam antibiotics 1 2 and for their possible new uses in synthetic organic chemistry 3-5 . PA has been identified as an N-terminal nucleophile hydrolase following specific catalytic and processing mechanisms 6-8 . During evolution the catalytic properties of enzymes have been optimized for their function in vivo. However their application as industrial biocatalysts often