title: Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G beta-lactamase.
abstract: Residue Arg220 was found to be important for the acylation of the Streptomyces albus G beta-lactamase by classical penicillins and cephalosporins bearing a carboxylate on C3 or C4. The R220L mutant exhibited strongly decreased kcat/Km values for those compounds. Conversely the acylation rates by benzylpenicillin methylester and deacetylcephalosporin C lactone were little affected, indicating a direct or indirect role of that positively charged residue in the interaction of the enzyme cavity with the negative charge of the substrate. Surprisingly that residue is not conserved in all class A beta-lactamases but when it is not present it can be seen in the known tertiary structures that the guanidinium group of another arginine side chain (Arg244) is similarly positioned. The mutation affected the behaviour of the enzyme towards cephaloridine much less than towards cephalothin. This might represent an example of substrate-assisted catalysis where the disappearance of a positive charge on the enzyme is partly compensated by the presence of a similarly charged group on one of the substrate side chains. All the experimental results are nicely explained by computer-modelling of the enzyme-substrate interactions.
authors.affiliation: Centre d'Ingéniérie des Protéines, Université de Liège, Belgium.
authors.forename: F
authors.initials: F
authors.lastname: Jacob-Dubuisson
authors.forename: J
authors.initials: J
authors.lastname: Lamotte-Brasseur
authors.forename: O
authors.initials: O
authors.lastname: Dideberg
authors.forename: B
authors.initials: B
authors.lastname: Joris
authors.forename: J M
authors.initials: JM
authors.lastname: Frère
mesh_terms: D000215:Acylation; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics*; D002384:Catalysis; D002511:Cephalosporins / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010406:Penicillins / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D002511:Cephalosporins; D010406:Penicillins; D001618:beta-Lactamases