Brønsted Analysis of an Enzyme-Catalyzed Pseudo-Deglycosylation Reaction: Mechanism of Desialylation in Sialidases

Abstract

The Micromonospora viridifaciens Y370G inverting mutant sialidase has been found to possess beta-sialidase activity with various fluoro-substituted phenyl beta-sialosides. A reagent panel of seven mono- and difluorophenyl beta-d-sialosides was synthesized, and these compounds were used, in conjunction with the parent phenyl beta-d-sialoside, to probe the mechanism of M. viridifaciens Y370G mutant sialidase-catalyzed hydrolyses. These hydrolysis reactions mimic the deglycosylation reaction step of the crucial tyrosinyl enzyme-bound intermediate that is formed during the corresponding wild-type sialidase reactions. The derived Brønsted parameter (beta(lg)) on k(cat)/K(m) is -0.46 +/- 0.02 for the four substrates that display significant activity, and these span a range of leaving group abilities (as judged by the pK(a) of their conjugate acids being between 7.09 and 9.87). The 4-fluoro, 2,3- and 2,5-difluorosubstrates display a diminished activity, whereas the 3,5-difluoro compound undergoes catalyzed hydrolysis exceedingly slowly. These observations, taken with solvent deuterium kinetic isotope effects (k(H(2))(O)/k(D(2))(O)) on the catalyzed hydrolysis of the 2-fluorophenyl substrate of 0.88 +/- 0.24 (k(cat)/K(m)) and 1.16 +/- 0.12 (k(cat)) and the poor inhibition shown by phenol (IC(50) > 1 mM), are consistent with glycosidic C-O cleavage being rate determining for both k(cat)/K(m) and k(cat) with little or no protonation of the departing aryloxide leaving group. The kinetic data reported herein are consistent with rate-limiting glycoside hydrolysis occurring via two distinct transition states that incorporates a nonproductive binding component for the tighter binding substrates.

Neuraminidase Substrate Promiscuity Permits a Mutant Micromonospora viridifaciens Enzyme To Synthesize Artificial Carbohydrates

“…For example, had we detected an estimated 10% of the 2,3-isomer in the presence of the 2,6-isomer as reaction products (Figures S7 and S8a, Supporting Information) and used the Haldane equation to calculate a free energy difference between the two regioisomers of >9 kJ mol –1 . However, in a previous report, we suggested that Mv NA Y370G-mediated catalysis proceeds via a transition state that involves no general acid catalysis (Figure c), a situation that occurs because the catalytic glutamate is likely to be deprotonated in the mutant enzyme ground state . In contrast, the transition state for the Mv NA-catalyzed glycosylation has an addition proton that bridges between the nucleophilic tyrosine and the general base glutamate , (Figure a).…”

“…Pyridine and acetic acid were purchased from Anachemia, and sodium acetate and magnesium chloride were purchased from Caledon Laboratories Ltd. All other chemicals, reagents, and enzymes were purchased from Sigma-Aldrich. Ph-βNeu5Ac and 2FPh-βNeu5Ac were synthesized according to literature procedures. , …”

“…21 As a first step toward increasing the yield of coupled products using mutant neuraminidases, we aimed to understand the mechanism of Y370G mutant-catalyzed reactions and then apply that knowledge toward the selection of optimal conditions for trans-glycosylation. 23 Since both academic and industry researchers express proteins using nonmammalian cell systems and recombinant DNA techniques, 24 an approach that produces nonsialylated glycoproteins, we decided to test the ability of our Y370G mutant sialidase to transfer N-acetylneuraminyl units onto a variety of carbohydrates. This paper presents a study of the range of acceptor sugars that undergo MvNA Y370G mutantcatalyzed coupling with N-acetylneuraminic acid and the identity of the resulting reaction products.…”

α-Thioglycoligase-based synthesis of O-aryl α-glycosides as chromogenic substrates for α-glycosidases

“…The aryl glycosides bearing different leaving groups are chromogenic substrates that can be used for detailed kinetic analysis of retaining glycosides [2][3][4]. The kinetic parameters of YicI were measured for the hydrolysis of the aryl ␣-xyloside substrates.…”

“…In addition, synthetic O-aryl glycosides with chromogenic or fluorogenic aglycons are invaluable substrates for the study of enzyme kinetics [2][3][4]. Therefore, the chemical synthesis of O-aryl glycosides is an interesting topic in carbohydrate chemistry.…”