A hydrogen-bonding network in mammalian sorbitol dehydrogenase stabilizes the tetrameric state and is essential for the catalytic power

Abstract

Subunit interaction in sorbitol dehydrogenase (SDH) has been studied with in vitro and in silico methods identifying a vital hydrogen-bonding network, which is strictly conserved among mammalian SDH proteins. Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. The results focus on a network of coupled hydrogen bonds in wild-type SDH that uphold the protein interface, which is specific and favorable to electrostatic, van der Waals and hydrogen-bond interactions between subunits, interactions that are crucial for the catalytic power of SDH.

X-ray crystal structure and small-angle X-ray scattering of sheep liver sorbitol dehydrogenase

“…Our structure does not include the coenzyme NAD. Earlier biochemical studies on SDH have shown it to only be functional when present in a tetrameric quarternary structure (Hellgren et al, 2007). The crystal structure that we report offers details of the tetramer interactions and shows the proximity of the substrate-binding pocket to the tetramer interface.…”

“…The conserved tetramer fits best to the solution tetramer structure observed in the SAXS experiment. Previous mutation studies have shown that an intact tetramer is crucial for biological activity (Hellgren et al, 2007). As seen in Fig.…”

“…The side chain of Tyr298 is spatially surrounded by the side chains of Asn300, Tyr109, Gln93 (Glu in hSDH) and Tyr139, which interact with each other by a hydrogen-bond network. This network must have been destabilized when Tyr109 was mutated to Phe, causing disruption of the dimer-dimer interface and a reduction in activity (Hellgren et al, 2007).…”

“…The change from a tetramer to a higher oligomer at around pH 10.0 corresponds to the pK a of a tyrosine residue side-chain hydroxyl. Mutation studies (Hellgren et al, 2007) had indicated Tyr110 as being crucial for tetramer stability. It is possible that once the tetramer falls apart the monomers might associate in a nonspecific manner to form larger clusters.…”

How weak an acid can be? Variations of H-bond and/or van der Waals Interaction of Weak Acids

“…By the middle the 20 th century, the discovery and atomic level description of natural products and bio-macromolecules (e.g. DNA, proteins) the significance of hydrogen bond formation emerged due to the fundamental role in the living systems [2]. Based on the X-ray diffraction (Photo 51) of DNA recorded by Rosaline Franklin, discovered not only that the DNA double helix hold together by H-bonds (and π-π stacking), but also that both replication, transcription and translation are driven by correct H-bond pairing mechanisms [3].…”