Evidence for a sialosyl cation transition‐state complex in the reaction of sialidase from influenza virus

Abstract

The enzyme mechanism of sialidase from influenza virus has been investigated by kinetic isotope methods, NMR, and a molecular dynamics simulation of the enzyme-substrate complex. Comparison of the reaction rates obtained with the synthetic substrate 4-methylumbelliferyl-N-acetyl-a-~-neuraminic acid and the [3,3-'H]-substituted substrate revealed /!I-deuterium isotope effects for V/Km ranging over 1.09 -1 .I 5 in the pH range 6.0 -9.5, whereas the effects observed for V in this pH range increased from 0.979 to 1.07. In D'O, ODV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while ODV was unchanged. Solvent isotope effects of 1.74 were obtained for both ODV/Km and ODV at pD 9.5, with ODV/K, decreasing and ODV remaining constant at acidic pD. 'H-NMR experiments confirmed that the initial product of the reaction is the a-anomer of N-acetyl-oneuraminic acid. Molecular dynamics studies identified a water molecule in the crystal structure of the sialidase-N-acetyl-D-neuraminic acid complex which is hydrogen-bonded to Asp1 51 and is available to act as a proton donor source in the enzyme reaction. The results of this study lead us to propose a mechanism for the solvent-mediated hydrolysis of substrate by sialidase that requires the formation of an endocyclic sialosyl cation transition-state intermediate.Sialidases catalyse the hydrolysis of terminal sialic acids from a range of glycoprotein, glycolipid and oligosaccharide substrates [I]. A large number of important biological functions are associated with the actions of this class of enzyme, including receptor recognition and masking, antigenic expression, protein degradation and some infectious processes [2]. Although a great deal of structural information has been gathered from crystallographic studies of influenza virus sialidase [3,4] and from sequence studies of bacterial sialidases [5, 61, there is a lack of detailed mechanistic information. Our recent interest in the biochemistry of sialidase from influenza virus [7, 81, has prompted us to undertake a mechanistic study of this enzyme.Isotope effects have been successfully used to show that some glycosidase-catalysed reactions occur with glycosyl cation-like transition states [9-131. However the application of isotope effects to a mechanistic study of sialidase has not been previously reported. The present study reports p-deuterium and solvent isotope effects on the enzyme reaction of sialidase from influenza virus, as well as the pH dependence and mul- V, secondary P-deuterium isotope effect on V.Enzyme. Sialidase (EC 3.2.1.18).tiple isotope effects of these deuterium probes. The enzyme reaction was also investigated by 'H-NMR spectroscopy to determine the stereochemistry around the anomeric centre of the released product, N-acetyl-D-neuraminic acid (NeuSAc). The kinetic results have been related to a molecular dynamics simulation of the sialidase-N-acetylneuraminic-acid complex, enabling us to propose a more detailed mechanism for the action of sialidase from influenza virus. MATERIALS A...

Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus

“…This effect was also observed by Chong et al [7] and is consistent with the proposal that influenza virus sialidase-catalysed hydrolysis of Neu5Aca2MU proceeds with substantial positive charge development at the reaction centre in the transition state for the formation of the sialosyl cation-enzyme intermediate. [7] ( Fig. 1).…”

“…Sialidase activity, using Neu5Ace2MU as substrate, was assayed by a modification [7] of a previously reported fluorometric method [11]. The fluorescence measured was linear with respect to the concentration of 4-methylumbelliferone.…”

“…The present study describes significant differences observed in isotope effects for this enzyme depending on the type of substrate used in the study. Chong et al [7] observed a significant inverse isotope effect on Vma x at pH 6.0, using the substrate 4-methylumbelliferyl-N-acetyl-~-D-neuraminic acid, indicating that hydrolysis of substrate by sialidase requires the formation of a sialosyl cation-enzyme transition state intermediate. The absence of such an effect observed by Guo et al [8] using p-nitrophenyl-N-acetyl-Ct-D-neuraminic acid as the substrate suggested, on the other hand, that the sialidase-catalysed reaction may equally well occur via hydrolysis of an ~-lactone intermediate.…”

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…The enzymatic reaction of rN2 and rN2-Tyr406Asp was further monitored with 1 H NMR using the avian influenza receptor analogue 3 0 -sialyllactose. According to previous studies confirming that influenza NA is a retaining glycosidase 13 , wild-type rN2 initially produced a-Neu5Ac, which mutarotates to form the more stable b-Neu5Ac over time. Interestingly, the b-anomer of Neu5Ac was detected as the initial product of 3 0 -sialyllactose after incubation with rN2-Tyr406Asp, which demonstrates that rN2-Tyr406Asp has switched from a retaining to inverting glycosidase mechanism ( Fig.…”

“…All of these current NA inhibitors were designed based upon the structure of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), a putative NA transition-state analogue 8,[10][11][12] . Insight into the NA catalytic mechanism and the proposed oxocarbenium ion transition-state intermediate were crucial for the design of Neu5Ac2en analogues with potent NA inhibitory activity 13,14 . However, evidence regarding the key influenza NA catalytic residues has remained elusive and the successful design of highly effective next-generation influenza NA inhibitors has proven to be a difficult task.…”