The design of mechanism-based enzyme inactivators to generate chemical probes for biological research is an important challenge in carbohydrate chemistry. Here we describe the synthesis and biological evaluation of a novel carbocyclic mechanism-based inactivator of galactosidases (glycoside hydrolase families 27 and 36). Upon catalysis of this unnatural substrate, a transient non-classical carbocation forms within the enzyme active site. We show that the inactivation event, which proceeds via a bicyclobutonium ion intermediate, leads to a single alkylation event that occurs on the enzymatic nucleophile, an aspartic acid residue in this case. We also show that the catalytic proficiencies for enzymatic hydrolysis of substrates and inactivation by our bicyclo[4.1.0]heptyl analogue of galactose differ by only a factor of 20. This inactivator has the potential for further development as a useful biological research tool for both basic research and biotechnological applications.
The MelA gene from Citrobacter freundii, which encodes a glycosyl hydrolase family 4 (GH4) α-galactosidase, has been cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the hydrolysis of phenyl α-galactosides via a redox elimination-addition mechanism involving oxidation of the hydroxyl group at C-3 and elimination of phenol across the C-1-C-2 bond to give an enzyme-bound glycal intermediate. For optimal activity, the MelA enzyme requires two cofactors, NAD(+) and Mn(2+), and the addition of a reducing agent, such as mercaptoethanol. To delineate the mechanism of action for this GH4 enzyme, we measured leaving group effects, and the derived β(lg) values on V and V/K are indistinguishable from zero (-0.01 ± 0.02 and 0.02 ± 0.04, respectively). Deuterium kinetic isotope effects (KIEs) were measured for the weakly activated substrate phenyl α-D-galactopyranoside in which isotopic substitution was incorporated at C-1, C-2, or C-3. KIEs of 1.06 ± 0.07, 0.91 ± 0.04, and 1.02 ± 0.06 were measured on V for the 1-(2)H, 2-(2)H, and 3-(2)H isotopic substrates, respectively. The corresponding values on V/K were 1.13 ± 0.07, 1.74 ± 0.06, and 1.74 ± 0.05, respectively. To determine if the KIEs report on a single step or on a virtual transition state, we measured KIEs using doubly deuterated substrates. The measured (D)V/K KIEs for MelA-catalyzed hydrolysis of phenyl α-D-galactopyranoside on the dideuterated substrates, (D)V/K((3-D)/(2-D,3-D)) and (D)V/K((2-D)/(2-D,3-D)), are 1.71 ± 0.12 and 1.71 ± 0.13, respectively. In addition, the corresponding values on V, (D)V((3-D)/(2-D,3-D)) and (D)V((2-D)/(2-D,3-D)), are 0.91 ± 0.06 and 1.01 ± 0.06, respectively. These observations are consistent with oxidation at C-3, which occurs via the transfer of a hydride to the on-board NAD(+), being concerted with proton removal at C-2 and the fact that this step is the first irreversible step for the MelA α-galactosidase-catalyzed reactions of aryl substrates. In addition, the rate-limiting step for V(max) must come after this irreversible step in the reaction mechanism.
The Y370G inverting mutant sialidase from Micromonospora viridifaciens possesses beta-sialidase activity with phenyl beta-sialoside (Ph-betaNeuAc) to give alpha-sialic acid as the first formed product. The derived catalytic rate constants for k(cat) and k(cat)/K(m) are 13.3 +/- 0.3 and (2.9 +/- 0.3) x 10(5) M(-)(1) s(-)(1), respectively. This enzyme is highly specific for the phenyl substrate, with substituted phenyl and thiophenyl leaving groups having k(cat) values that are at least 1000-fold lower. In addition, the Y370G mutant can transfer the sialic acid moiety from Ph-betaNeuAc to lactose in yields of up to 13%. Greater than 90% of the sialyl-lactose product formed in the coupling reactions is the alpha-2,6-isomer. A library encoding 6 x 10(5) different sialidases was constructed by mutating Y370, E260, T309, N310, and N311, residues that include and are proximal the catalytic tyrosine residue. A total of 2628 individuals were screened for hydrolytic activity against 4-nitrophenyl 2-thio-beta-sialoside and 4-methylumbelliferyl beta-sialoside. However, none of the mutants screened possessed a significant activity against either of the beta-sialosides.
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.
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