Different transition-state structures for the reactions of beta-lactams and analogous beta-sultams with serine beta-lactamases.
Beta-sultams are the sulfonyl analogues of beta-lactams, and N-acyl beta-sultams are novel inactivators of the class C beta-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C-O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl beta-lactams are substrates for beta-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation-deacylation process. The rates of acylation of the enzyme by the beta-lactams, measured by the second-order rate constant for hydrolysis, kcat/K(m), and those of sulfonylation by the beta-sultams, measured by the second-order rate constant for inactivation, k(i), both show a similar pH dependence to that exhibited by the beta-lactamase-catalyzed hydrolysis of beta-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl beta-lactams increase the rate of alkaline hydrolysis and give a Bronsted beta(lg) of -0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted beta(lg) for the beta-lactamase-catalyzed hydrolysis of the same substrates is -0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted beta(lg) for the alkaline hydrolysis of N-aroyl beta-sultams is -0.73, similar to that for the beta-lactams, that for the sulfonylation of beta-lactamase by these compounds is -1.46, compatible with significant amide anion expulsion/S-N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the beta-sultam.
Tsang
Wing Y
WY
Department of Chemical and Biological Sciences, The University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK.
Ahmed
Naveed
N
Hinchliffe
Paul S
PS
Wood
J Matthew
JM
Harding
Lindsay P
LP
Laws
Andrew P
AP
Page
Michael I
MI
D000215:Acylation; D001665:Binding Sites; D016972:Enterobacter cloacae / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*; D047090:beta-Lactams / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
D013449:Sulfonamides; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D001618:beta-Lactamases