Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Monday Speaker Abstracts

PROTEIN ELECTRIC FIELDS REGULATE COVALENT INHIBITION OF BETA LACTAMASES Steven G. Boxer ; Zhe Ji 1 ; 1 Stanford University, Chemistry, Stanford, CA, USA Beta-lactamases can use their protein machinery to hydrolyze some beta-lactam antibiotics rapidly, yet are less proficient towards other substrates, and are even trapped by efficient inhibitors. We sought to understand how covalent inhibitors function by studying the physical basis for their differences in reactivity from substrates, using TEM-1 as a model beta-lactamase. While penicillin G, a b-lactam substrate, is subject to a two-step hydrolysis mechanism, enzyme acylation and hydrolytic deacylation, avibactam as a covalent inhibitor can perform rapid acylation but sluggish deacylation, trapping many b-lactamase targets in the inactive acyl enzyme state. We examine the different reactivities of penicillin G and avibactam under the framework of electrostatic catalysis. Electric fields projected onto a bond involving charge displacement can stabilize its transition state and therefore enhance the rate. Using the vibrational Stark effect to quantify the magnitude of electric fields, we observed that C=O in avibactam, the key bond undergoing reactions, experiences high electric fields as that in penicillin G does in the Michaelis complex (see Kozuch poster), but contrastingly lower fields in the acyl-enzyme, consistent with the observation of fast acylation and slow deacylation. These electric fields are mainly exerted by hydrogen bonds between the avibactam C=O and protein backbone amides. By replacing a backbone amide with an ester using amber suppression, we quantified the role of the hydrogen bond in exerting electric fields and accelerating reactions. Compared with penicillin G, avibactam’s C=O experiences a lower electric field by 67 MV/cm when passaging towards deacylation, leading to 10 6 -fold rate diminution. Our studies provide physical insights into the long residence time of covalent inhibitors—electrostatic stabilization can contribute more than intrinsic bond stability. We envision that active site electric fields can act as a general, quantitative descriptor to guide the design of covalent drugs.

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