1H-NMR-Untersuchungen zur Mutarotation derN-Acetyl-D-neuraminsäure

Friebolin, H.; Supp, Martin; Brossmer, Reinhard; Keilich, G.; Ziegler, Dietmar

doi:10.1002/ange.19800920315

Cited by 15 publications

(9 citation statements)

References 4 publications

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“…This is in accord with the proposal, based on observations made with bacterial sialidase, that retention of configuration is a general property of sialidases [35]. The calculated mutarotation rates are in good agreement with those obtained by other workers [23, 241.…”

Section: -supporting

confidence: 92%

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

Chong

Pegg

Taylor

et al. 1992

European Journal of Biochemistry

182

155

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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...

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Section: -supporting

confidence: 92%

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

Chong

Pegg

Taylor

et al. 1992

European Journal of Biochemistry

182

155

View full text Add to dashboard Cite

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“…The general findings of this study and others [24][25][26][27][28][29][30] suggest that irrespective of the source of the enzyme all sialidases appear to be retaining enzymes.…”

Section: +supporting

confidence: 76%

“…All of the carbohydrates evaluated are either known inhibitors of other sialidases or are potential sialidase inhibitors. Finally, we [24][25][26][27] and others [28,29] have explored the catalytic reaction of sialidases from various sources by 1 H NMR spectroscopy. In light of these previous studies, we have taken the opportunity to further characterise the sialidase activity from cytosolic skeletal muscle by using 1 H NMR spectroscopy to monitor the hydrolysis of a synthetic sialoside.…”

Section: Introductionmentioning

confidence: 99%

An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

et al. 2005

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Rat cytosolic sialidase is expressed at elevated levels in skeletal muscle and is believed to play a role in the myogenic differentiation of muscle cells. Here, we observed varying levels of enhancement of sialidase activity in the presence a range of divalent cations. In particular, a significant enhancement of activity was observed in the presence of Ca 2+ . Conversely, inhibition of the sialidase activity was found when the enzyme was incubated in the presence of Cu 2+ , EDTA, and a range of carbohydrate-based inhibitors. Finally, an investigation of the enzymatic hydrolysis of a synthetic substrate, 4-methylumbelliferyl N-acetyl-a-D D-neuraminide, by 1 H NMR spectroscopy revealed that the reaction catalysed by rat skeletal muscle cytosolic sialidase proceeds with overall retention of anomeric configuration. This result further supports the notion that all sialidases appear to be retaining enzymes.

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“…NMR studies were performed with the products in order to determine the stereochemical outcome of mutant‐catalyzed hydrolysis of MUαNeu5Ac (Table 1). Given that the half‐time for sialic acid mutarotation under the reaction conditions is approximately 80 min,9, 10 it is impossible to confirm whether the hydrolysis reaction of Y370H proceeds by inversion or retention of the anomeric configuration. Indeed, it is also impossible to determine the stereochemical outcome of the hydrolysis of substrates such as sialyl lactose that are turned‐over slowly.…”

Section: Resultsmentioning

confidence: 99%

Two Nucleophilic Mutants of the Micromonospora viridifaciens Sialidase Operate with Retention of Configuration by Two Different Mechanisms

Watson¹,

Newstead²,

Narine³

et al. 2005

ChemBioChem

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Mutants of the Micromonospora viridifaciens sialidase, Y370E and Y370F, are catalytically active retaining enzymes that operate by different mechanisms. Previous substitutions with smaller amino acids, including Y370D, yielded inverting sialidases. At least one water molecule can fit into the active-site cavity of this mutant and act as a nucleophile from the face opposite the leaving group (Biochemistry 2003, 42, 12 682). Thus, addition of a CH(2) unit (Asp versus Glu) changes the mechanism from inversion back to retention of configuration. Based on Brønsted beta(lg) values, it is proposed that the Y370E mutant reacts by a double-displacement mechanism (beta(lg) on k(cat)/K(m) -0.36+/-0.04) with Glu370 acting as the nucleophile. However, the Y370F mutant (beta(lg) on k(cat)/K(m) -0.79+/-0.12) reacts via a dissociative transition state. The crystal structure of the Y370F mutant complexed with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid shows no significant active-site perturbation relative to the wild-type enzyme.

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1H-NMR-Untersuchungen zur Mutarotation derN-Acetyl-D-neuraminsäure

Cited by 15 publications

References 4 publications

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

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

An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

Two Nucleophilic Mutants of the Micromonospora viridifaciens Sialidase Operate with Retention of Configuration by Two Different Mechanisms

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