Amidases Have a Hydrogen Bond that Facilitates Nitrogen Inversion, but Esterases Have Not

Abstract

The fact that proteases/amidases can hydrolyze amides efficiently whereas esterases can not has been discussed during the last decades. By using molecular modeling we have found a hydrogen bond in the transition state for protease/amidase catalyzed hydrolysis of peptides and amides donated by the scissile NH‐group of the substrate. The hydrogen‐bond acceptor was found either in the enzyme (enzyme assisted) or in the substrate (substrate assisted). This new interaction with the NH‐hydrogen in the transition state (TS) was found in sixteen proteases/amidases, which represent ten different reaction mechanisms and eleven different folding families. Esterases lack this interaction and, therefore, they are slow in hydrolyzing amides. By mimicking the substrate‐assisted catalysis found in amidases we were able to shift reaction specificity of amide over ester synthesis of Candida antarctica lipase B one hundred fold. We propose that the hydrogen bond facilitates nitrogen inversion in amidases.

Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

“…Left: Boosting promiscuous activity by focusing on the in silico discovery [23,24] and engineering [34] of key mechanistic structural elements in modern enzymes is represented by the forward gear. This is exemplified by focusing on a hydrogen bond acceptor (arrow) that could act as a shifter for enhanced amidase over esterase activity by facilitating nitrogen inversion (structure shown), a key step in amide bond hydrolysis [26]. We have shown that the previously unacknowledged key hydrogen bond donated by the reacting NH-group facilitates nitrogen inversion, the overall rate limiting step of enzyme catalyzed amide bond hydrolysis [26,27,35].…”

“…This is because proteases and amidases, which cleave both esters and amides with high efficiency (relative activity 10 3 due to the resonance stabilization of amides [25]), capitalize on the additional hydrogen sitting on the reacting nitrogen atom of amide substrates. Amidases afford hydrogen bond formation to the reacting NH-group in the transition state (TS) [26], which results in efficient amidase activity ( Figure 1, left), regardless of protein fold, reaction mechanism, or topology of catalytic dyads/triads [27] displayed by amidases [28].…”

“…In contrast, esterases have been found to lack an enzyme-assisted hydrogen bond acceptor for this key interaction, which results in low or even absent promiscuous activities towards amide-based biochemistries [26]. Inspired by the recently acquired detailed mechanistic knowledge of these at first glimpse analogous chemistries, and in an effort to shed additional light on their possible evolution, which still remains elusive [29,30], we undertook a simplistic approach aiming for the discovery of key mechanistic interactions in esterases that could serve as a specificity shifter.…”

“…In another study, the reconstruction of extinct β-lactamases afforded highly thermostable scaffolds [32]. As we were previously unable to identify enzyme-assisted hydrogen bond acceptors in esterases belonging to the α,β-hydrolases [26], we reasoned that analyzing alternative and existing enzyme folds would be a relevant second strategy for the identification of promiscuous esterase scaffolds that could serve as a starting point for further engineering (represented by the forward gear, Figure 1, left). Capitalizing on a previously calculated transition state structure of nitrogen inversion [29], in silico analysis readily identified an enzyme-assisted hydrogen bond acceptor in patatin, an esterase from potato with a unique Ser/Asp catalytic dyad that does not display an α,β-hydrolase fold [33].…”

“…This is exemplified by focusing on a hydrogen bond acceptor (arrow) that could act as a shifter for enhanced amidase over esterase activity by facilitating nitrogen inversion (structure shown), a key step in amide bond hydrolysis [26]. We have shown that the previously unacknowledged key hydrogen bond donated by the reacting NH-group facilitates nitrogen inversion, the overall rate limiting step of enzyme catalyzed amide bond hydrolysis [26,27,35]. As the specific orientation of the single lone pair of the reacting nitrogen is governed by stereoelectronic effects [36], nitrogen inversion is required during biocatalysis for the generation of a productive second tetrahedral intermediate.…”