Combined QM/MM mechanistic study of the acylation process in furin complexed with the H5N1 avian influenza virus hemagglutinin's cleavage site

Abstract

Combined quantum mechanical/molecular mechanical (QM/MM) techniques have been applied to investigate the detailed reaction mechanism of the first step of the acylation process by furin in which the cleavage site of the highly pathogenic avian influenza virus subtype H5N1 (HPH5) acts as its substrate. The energy profile shows a simultaneous mechanism, known as a concerted reaction, of the two subprocesses: the proton transfer from Ser368 to His194 and the nucleophilic attack on the carbonyl carbon of the scissile peptide of the HPH5 cleavage site with a formation of tetrahedral intermediate (INT). The calculated energy barrier for this reaction is 16.2 kcal.mol(-1) at QM/MM B3LYP/6-31+G*//PM3-CHARMM22 level of theory. Once the reaction proceeds, the ordering of the electrostatic stabilization by protein environment is of the enzyme-substrate < transition state < INT complexes. Asp153 was found to play the most important role in the enzymatic reaction by providing the highest degree of intermediate complex stabilization. In addition, the negatively charged carbonyl oxygen of INT is well stabilized by the oxyanion hole constructed by Asn295's carboxamide and Ser368's backbone.

Mechanisms of Proteolytic Enzymes and Their Inhibition in QM/MM Studies

“…Apparently, the reaction mechanism of furin differs from the trypsin-like proteases, as cleavage of the H5N1 avian influenza virus hemagglutinin involves a single transition state in a concerted reaction, with the H + transfer from Ser368 to His194 together with the nucleophilic attack of the Ser368 Oγ on the carbonyl C of the scissile bond [162]. A different underlying mechanism proceeds in the transesterification of L-phenylalanine-Nacetyl-ethyl-ester, which is attacked by the Ser221 Oγ nucleophile of subtilisin Carlsberg with a concerted proton transfer to His64 [163].…”

Why All the Fury over Furin?

“…In furin, Asp153 is H-bonded with His194, probably increasing the p K a of its imidazole nitrogen and thus allowing His194 to act as a powerful general base that activates the Ser368 nucleophile. 74 , 196 − 198 An oxyanion hole formed by the side chain Asn295 and the backbone amino of Ser368 stabilizes the charge build-up on the tetrahedral intermediates. 74 , 196 , 197 The histidine base aids the first leaving group by donating a proton and also activates the hydrolytic water molecule by abstracting a proton as the remaining OH – attacks the acyl-enzyme intermediate ( Figure 6 ).…”

“… 74 , 196 − 198 An oxyanion hole formed by the side chain Asn295 and the backbone amino of Ser368 stabilizes the charge build-up on the tetrahedral intermediates. 74 , 196 , 197 The histidine base aids the first leaving group by donating a proton and also activates the hydrolytic water molecule by abstracting a proton as the remaining OH – attacks the acyl-enzyme intermediate ( Figure 6 ). The chemical reaction catalyzed by furin normally involves limited conformational changes at the active site residues, unlikely to perturb remote substrate-binding subsites of the enzyme.…”

Computational Studies of Influenza A Virus at Three Important Targets: Hemagglutinin, Neuraminidase and M2 Protein

“…Another study of HA enzymatic reaction mechanism was conducted using the hybrid quantum mechanics/molecular mechanics (QM/MM) [17]. This particular approach was employed to investigate the first step of the acylation process in furin where its substrate was the cleavage site of the HPIV subtype H5N1.…”