Inhibition of β-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new β-lactam antibiotic

Abstract

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.

Effect of an Amino Acid Insertion into the Omega Loop Region of a Class C β-Lactamase on Its Substrate Specificity

“…Substrate hydrolysis was measured as the change in A 265 or A 290 for cephalothin, A 260 and A 330 for cefuroxime, A 260 for ceftazidime, A 318 for aztreonam, A 235 for benzylpenicillin, or A 230 for ampicillin with a temperature-controlled 220A spectrophotometer (Hitachi). The k cat and K m values were determined from the reaction curve by fitting to the integrated form of the Lineweaver-Burk plot (12). Because the K m values of some mutants for ceftazidime and cefuroxime were so high that the reaction could not be saturated, 100 µM ceftazidime and 500 µM cefuroxime were used to determine the k cat and K m values, and the k cat /K m ratio was determined from measurements of the first-order rate of hydrolysis with substrate concentrations much less than the K m values deduced with the on-line method.…”

Inhibition of the RTEM .beta.-lactamase from Escherichia coli. Interaction of the enzyme with derivatives of olivanic acid

“…The reasonable nucleophilicity of a sulfate oxygen (Backer & Dubsky, 1920), the formation of a six-membered ring, and the resulting tetrahedral adduct (which, by analogy with the "oxyanion hole" of the serine proteases, may be especially well stabilized by the enzyme) would all be consistent with this suggestion. It is true, however, that some carbapenems that lack a sulfate ester at C-8, such as PS-5 (Okamura et al, 1980;Fukagawa et al, 1980) and 7V-formimidoylthienamycin (Kahan et al, 1979), show kinetic behavior analogous to that of 2 (C. Kemal, unpublished results). While the differing stereochemistry at C-6 and at C-8 in the thienamycin series could be important, the addition of sulfate to the acyl-enzyme carbonyl group is clearly inadequate to accommodate the behavior of all carbapenems with the 0-lactamase.…”

Characterization of eight beta-lactamases of Gram-negative bacteria

“…The discrepancy may be attributed to the difference in the assay method employed. We employed an acidimetric method with a pH stat because this method is applicable to a wide range of 3-lactam antibiotics and a computer program for calculating Km (4).…”