Mechanism and Catalytic Site Atlas

Introduction

GAPN uses an ordered sequential two-step mechanism for catalysis - acylation followed by deacylation. First, NADP+ binds to the enzyme, inducing a conformational change in the active site, resulting in the activation of a competent enzyme. The positive charge of the nicotinamide ring of NADP+, the amide peptide nitrogens of residues Cys284 and Thr285, and possibly the involvement of other structural elements stabilise the thiolate form of Cys284. G3P binding leads to formation of a thiohemiacetal intermediate via a nucleophilic attack by the Cys284 thiolate. This is followed by an oxidoreduction process in which a hydride is transferred from the intermediate to the nicotinamide ring of NADP+ forming a thioacylenzyme intermediate. This is able to take place without catalytic assistance due to the presence of an oxianion hole, formed by Asn154 and the amide nitrogen of Cys284, interacting with the C1 oxygen of the substrate, keeping the oxygen deprotonated. The oxyanion hole is also involved in the stabilisation of the tetrahedral thioacyl intermediate. Then deacylation occurs, where Glu250 orientates and activates water which nucleophilically attacks the C1 carbon of the thioacyl intermediate, leading to an acid product and, finally, the release of the reduced cofactor (NADPH). The isomerisation and movement of the nicotinamide ring of NADPH after the oxidoreduction stage is required to allow Glu250 to perform its role in deacylation.