Serine Dehydratase - Enzyme Mechanism

Enzyme Mechanism

The degradation of serine to pyruvate is an example of a pyridoxal phosphate-dependent (PLP) catalyzed Beta-elimination reaction. Beta-eliminations mediated by PLP yields products that have undergone a two-electron oxidation at C-alpha. In general, beta-eliminations involve the removal of a halide and a proton from the adjacent beta-carbon to give a double bond; thus, the origin of the double bond pi electrons are from the C-H bond on the beta carbon of the substrate.

Beta eliminations occur with no net oxidation or reduction of PLP. In overall terms, the reaction catalyzed by serine dehydratase involves two steps: catalytic elimination and a nonenzymatic hydrolysis reaction. The main role of SDH is to lower the activation energy of this reaction by binding the coenzyme and substrate in a particular conformational geometry.

Mechanistic Steps:

(In panel 1 of Figure 5) In the SDH enzyme’s active site, Lys41 is located above the PLP molecule with its R group NH₂ connected to C4 of PLP by a Schiff base linkage. The phosphate group of PLP is located in a pocket of G residues. Serine enters the active site and its positively charged amino group attracts the negatively charged phosphate group of PLP. The intermediate PLP-Ser aldimine is created. SDH's role is to orient the serine molecule’s Calpha-H parallel to the overlapping 2p orbitals of the PLP pi system; in other words, SDH holds serine perpendicular to the plane of the PLP ring. ( See Figure 6 for orientation of substrate with PLP).

(In panel 2 of Figure 5) The amino group of serine protonates the PLP phosphate by forming a H-bond. The deprotonated amino group of Serine is now a good nucleophile that attacks the Lys-PLP Schiff base at the C4 carbon (shown in panel 1). The Lys41 is released from PLP.

(In panel 3 of Figure 5) The COOH group of serine is positioned tightly in the SDH enzyme so that the serine molecule is perpendicular to the PLP pi system. The R group OH group participates in two hydrogen bonds with SDH’s Ala222 and the protonated phosphate of PLP. The protonated phosphate of PLP then acts as an acid and donates its proton to hydroxyl of serine. In a concerted fashion, the R group hydrogen of serine is removed by Lys41 and water is released. The intermediate created is PLP-aminoacrylate.

In the reaction as the water leaves from the Beta-carbon of the substrate, the SDH orients the newly created double bond perpendicular to the PLP plane (Figure 6). This allows the new pi bonds between Calpha and Cbeta to form resonance with the PLP pi system. (Figure 6)

(In panel 4 of Figure 5) Lys41 from SDH's active site attacks C4 of PLP, forming a tetrahedral intermediate.

(In panel 5 of Figure 5) A Schiff base linkage is then made and the aminoacrylate group is released as pyruvate.

(In panel 6 of Figure 5) The aminoacrylate released from PLP is unstable in aqueous solution and rapidly tautomerizes to the preferred imine form; this is spontaneously hydrolyzed to yield alpha-keto acid product of pyruvate. The aminoacrylate is nonezymatically deaminated to pyruvate by hydrolysis. The enzyme-PLP Schiff base linkage is reformed.

Figure 5 shows the mechanism of converting L-Serine into Pyruvate. The Figure displays the SDH active site, PLP coenzyme and substrate.

Figure 6 shows the role of SDH in orienting the PLP molecule perpendicular to the substrate Serine.