Inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme.
The pH-rate profile for inactivation of the RTEM-1 cysteine beta-lactamase by iodoacetate supports previous evidence [Knap & Pratt (1989) Proteins Struct. Funct. Genet. 6, 316-323] for the activation of the active-site thiol group by adjacent functional groups. The enhanced reactivity of iodoacetate, with respect to that of iodoacetamide, suggests the influence of a positive charge in the active site. The reactivity of iodoacetate is not affected by dissociation of an active-site functional group of pKa 6.7, which increases the reactivity of neutral reagents, probably because of a compensation phenomenon; it is, however, lost on dissociation of an acid of pKa 8.1. It is concluded that the active cysteine beta-lactamase has four functional groups at the active site, one nucleophilic thiolate of Cys-70, one neutral acid (most probably the carboxy group of Glu-166, from the crystal structures) and two cationic residues (most probably Lys-73 and Lys-234). A comparison of these results with the pH-dependence of reactivity of the native RTEM-2 beta-lactamase suggests that the active form of the latter enzyme is also monocationic, although the nucleophile (Ser-70) is likely to be neutral in this case and the carboxylic acid dissociated. A mechanism of class A beta-lactamase catalysis is discussed where the Glu-166 carboxylate acts as a general base/acid catalyst and Lys-73 is principally required for electrostatic stabilization of the anionic tetrahedral intermediate.
Knap
A K
AK
Department of Chemistry, Wesleyan University, Middletown, CT 06457.
Pratt
R F
RF
D001665:Binding Sites; D001888:Boric Acids / Q000494:pharmacology; D002384:Catalysis; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007461:Iodoacetates / Q000494:pharmacology*; D019807:Iodoacetic Acid; D010400:Penicillin G / Q000378:metabolism; D010957:Plasmids; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001888:Boric Acids; D007461:Iodoacetates; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; C056342:beta-lactamase TEM-2; D003545:Cysteine; D010400:Penicillin G; C032688:boric acid; D019807:Iodoacetic Acid