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Active Role of the Substrate During Catalysis by the Therapeutic Enzyme L‐Asparaginase II
Author(s) -
Rempe Susan,
Vanegas Juan,
Anishkin Andriy,
Rogers David,
Sukharev Sergei
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.573.51
Subject(s) - deamidation , chemistry , nucleophile , substrate (aquarium) , asparagine , active site , stereochemistry , oxyanion hole , enzyme , enzyme catalysis , catalysis , asparagine synthetase , amide , ab initio , biochemistry , organic chemistry , oceanography , geology
Bacterial type II L‐Asparaginases (ASPII) have been used for over four decades to treat acute lymphoblastic leukemia, yet a full reaction mechanism remains unknown. ASPII enzymes catalyze the deamidation of both asparagine (Asn) and glutamine (Gln), which results in the formation of aspartate (Asp) and glutamate (Glu) respectively, and the by‐product ammonia. Proposed ASPII mechanisms to date have yet to explain the absolute requirement of a substrate α‐carboxyl group, and clearly identify the role of the catalytic threonines T12 and T89. Here, we study the reaction mechanism of asparagine degradation by ASPII through ab initio molecular dynamics (MD) simulations. We select a reduced system from the substrate‐bound enzyme obtained by classical MD, and explore different initial reaction pathways by driving the system in steered simulations. Our results show that direct nucleophilic attack by T12 produces a highly unstable substrate‐enzyme intermediate, as the stabilization provided by the nearby protons (i.e., the “oxyanion hole”) is insufficient to sustain the high energy state. We find that the substrate‐enzyme intermediate can be stabilized by first protonating the substrate's amide oxygen through the K162‐T89 proton bridge. Furthermore, the α‐carboxyl of the substrate acts as a proton acceptor for the hydroxyl sidechain of T12 during nucleophilic attack. We conclude by showing that a complete deamidation mechanism may require a sequence of several nucleophilic attacks by both T12 and T89, with K162 playing a critical role as a proton buffer during the course of the reaction.