Kinetic and Inhibition Studies of Bacillus cereus β-Lactamase Using a Spectrophotometric Method

Abstract

The use of a Spectrophotometric method is reported for the characterization of a /Mactamase (EC 3.5.2.6) from Bacillus cereus. Absorption coefficients of the mercaptids of various penicillins were determined with this method. The enzyme was kinetically characterized using penicillins.Inhibition studies with Bacillus cereus 0-lactamase and various penicillins showed a substrate type of inhibition, indicating an additional binding site for substrates without catalytic activity.The dissociation constant of this binding site was determined and the influence of this binding site upon the catalytic activity is discussed. Studies with /J-lactamase-stable penicillins as inhibitors and various penicillins showed different types of inhibition, which indicated the presence of an additional catalytically inactive binding site. Experiments with clavulanic acid, a /Mactamase inhibitor without remarkable intrinsic antibacterial activity, showed a mixed type of inhibition. Based on the hypothesis for the existence of more than one substrate binding site on the enzyme, clavulanic acid was found to be bonded to both the catalytic active and the catalytic inactive binding site. Kinetische Untersuchungen und Hemmungsstudien an einer ß-Lactamase aus Bacillus cereus mit einer spektrophoto· metrischen MethodeZusammenfassung: Wir haben eine photometrische Methode zur Bestimmung von Penicillinen für die Charakterisierung einer ß-Lactamase (EC 3.5.2.6) ms Bacillus cereus angewendet und die molaren Absorptionskoeffizienten der aus verschiedenen Penicillinen gebildeten Mercaptide bestimmt. Das kinetische Verhalten des Enzyms wurde unter Verwendung verschiedener Penicilline überprüft. Untersuchungen mit hohen Substratkqnzentrationen ergaben für die ß-Lactamase aus Bacillus cereus mit verschiedenen spaltbaren Penicillinen das Bild einer Substratüberschußhemmung. Aufgrund dieses Hemmtyps wird neben der katalytisch aktiven Stelle eine weitere, substratbindende Stelle auf der untersuchten 0-Lactamase vermutet. Die Dissoziationskonstänte dieser Bindungsstelle wird bestimmt und der Einfluß dieser nicht katalytisch aktiven Stelle auf die katalytisch aktive Stelle diskutiert. Versuche mit jS-Lactamase-festen Penicillinen als Inhibitor und verschiedenen Penicillinen ergaben unterschiedliche Hemmtypen, die unter Einbeziehung der postulierten katalytisch inaktiven substratbindenden Stelle erklärt werden. Clavulansäure, ein ß-Lactamäse-Inhibitor ohne nennenswerte eigene antimik-robieUe Aktivität, zeigte das Bild eines gemischten Hemmtyps. Unter Zugrundelegung der Mehrbindungsstellenhypothese besetzt Clavulansäure sowohl die katalytisch aktive, als auch die katalytisch inaktive Bindungsstelle. , ible penicillin from enzymatic inactivation. The inhibi-In u on tion of a Bacillus cereus 0-lactamase by oxacillin, Since the discovery of peniciUinase in 1940 by Abraham methici y in ^d nafcillin has been described as com-& Chain (1) and the recognition of the importance of pe titive (6, 7), but other observations indicate that a 0-lactamases in the resistance o...

Purification and characterization of a new β-lactamase OXA-205 from Pseudomonas aeruginosa

“…Also, we obtained Hills coefficients (n = 2.1 ± 0.2 and x = 4.9 ± 0.7 for carbenicillin and n = 1.1 ± 0.2 and x = 3.6 ± 0.7 for oxacillin) which indicated cooperative effects in both the hydrolysis and inhibition by carbenicillin, while only inhibition by oxacillin demonstrated cooperative manner. To the best of our knowledge, substrate inhibition is not unique among β-lactamases, however it seems that it is the first example among non-metallo-β-lactamases [ 32 , 33 ]. Currently, the exact mechanism of substrate inhibition for β-lactamases is unknown but it was suggested that it might be linked to the bulky aromatic ring substitutions in some of the antibiotics and the peculiarities of the active sites of β-lactamases [ 33 ].…”

Exploiting a Bacterial Drug‐Resistance Mechanism: A Light‐Activated Construct for the Destruction of MRSA

“…β‐LEAP is designed in such a way that, upon cleavage by β‐lactamase (Scheme ), the PS is released from homodimeric, ground‐state quenching to yield an enzyme‐specific, light‐activated antimicrobial action. The β‐lactamases were ideal targets for this proof‐of‐principle study owing to the prevalence of β‐lactamase expression among bacteria and its high enzymatic efficiency 8. 9…”