Ser-796 of β-galactosidase (Escherichia coli) plays a key role in maintaining a balance between the opened and closed conformations of the catalytically important active site loop

A loop (residues 794–803) at the active site of β-galactosidase (Escherichia coli) opens and closes during catalysis. The α and β carbons of Ser-796 form a hydrophobic connection to Phe-601 when the loop is closed while a connection via two H-bonds with the Ser hydroxyl occurs with the loop open. β-Galactosidases with substitutions for Ser-796 were investigated. Replacement by Ala strongly stabilizes the closed conformation because of greater hydrophobicity and loss of H-bonding ability while replacement with Thr stabilizes the open form through hydrophobic interactions with its methyl group. Upon substitution with Asp much of the defined loop structure is lost. The different open-closed equilibria cause differences in the stabilities of the enzyme · substrate and enzyme · transition state complexes and of the covalent intermediate that affect the activation thermodynamics. With Ala, large changes of both the galactosylation (k2) and degalactosylation (k3) rates occur. With Thr and Asp, the k2 and k3 were not changed as much but large ΔH‡ and TΔS‡ changes showed that the substitutions caused mechanistic changes. Overall, the hydrophobic and H-bonding properties of Ser-796 result in interactions strong enough to stabilize the open or closed conformations of the loop but weak enough to allow loop movement during the reaction.

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► β-Galactosidase active site loop (residues 794–803) opens/closes during reaction.
► If loop is ‘open’ enzyme substrate complex stabilized.
► If loop is ‘closed’ transition states stabilized.
► Substituted enzyme structures show Ser-796 is important for open/closed equilibrium.
► Altered rates and thermodynamics show loop conformation is important for mechanism.