The mechanism of catalysis by L-asparaginase.

Abstract

Two bacterial type II L-asparaginases, from Escherichia coli and Dickeya chrysanthemi, have been playing a critical role for over 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by E. coli type II L-asparaginase (EcAII). Our model was developed based on new structural and biochemical experiments combined with previously published data. The proposed mechanism is supported by quantum chemistry calculations based on density functional theory (DFT). We provide strong evidence that EcAII catalyzes the reaction according to the double-displacement (ping-pong) mechanism, with formation of a covalent intermediate. Several steps of catalysis by EcAII are unique when compared to reactions catalyzed by other known hydrolytic enzymes. Here, the reaction is initiated by a weak nucleophile, threonine, without direct assistance of a general base, although a distant general base is identified. Furthermore, tetrahedral intermediates formed during catalytic process are stabilized by a never previously described motif. Although the scheme of the catalytic mechanism was developed based only on data obtained from EcAII and its variants, this novel mechanism of enzymatic hydrolysis could potentially apply to most (and possibly all) L-asparaginases.

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Identifiers

DOI

10.1021/acs.biochem.0c00116

Journal

Biochemistry

Year

2020