The NAD binding site of each subunit in GAPDH is located towards the core of the enzyme.6 Pairs of active sites are linked through a flexible polypeptide loop between the subunits.1 There are many hydrogen bonds that occur at the active site to help increase the stability of the enzyme once the NAD is bound.10 There are several residues within the active site that are important for the binding of NAD. One of the most important residues is Cys-149.7 The sulfhydryl group of this residue allows the hydride ion to leave during the oxidation step of the reaction.12 A modification of this residue also causes a conformational change in the active site.10 His-176 is also cataylitically essential in this active site.9 First, the N atom is hydrogen bonded to the mainchain carbonyl oxygen of Ser-177.
| This bond fixes the pIane of the imidazole ring and prevents its rotation.8 It is responsible for protonating the C1 oxygen of G3P.1 This residue also acts as a chemical activator to enhance the activity of Cys-149 thiol group. This activation occurs though the formation of an ion pair with the imidazolium ring of His-176 and Cys-149.9 This lowers the pka of the Cys-149 and allows it to be more nucleophilic. When NAD binds to the active site, it causes a contraction that brings Cys-149 and His-176 closer together. The tetrahedral intermediate that is formed during the reaction is also stabilized by the imidazole nitrogen of His-176 along with the carbonyl oxygen of the glyceraldehyde 3-phosphate.9 In fact, the second step of the overall |
| reaction requires the presence of Cys-149 and His-176. During the oxoreduction step, His-176 also functions as a base catalyst by facilitating the hydride transfer to the C4 position of the nicotinamidum ring of NAD.9 This is a very important part of the limiting step of the overall reaction.
There are also hydrophobic interactions that occur at both ends of the NAD molecule.6 The adenine ring of the NAD has hydrophobic interactions with Leu-33 |
There are two substrate anion binding sites as well as the NAD binding site in each subunit active site.3 One of these sites is the Ps , or substrate anion site, and the other is the Pi, or the inorganic phosphate site. The substrate aninon site is usually occupied by a sulfate ion that is hydrogen bonded to to the side chains of Arg-231 and Thr-179 as well as the nicotinamide ribose of NAD. The inorganic phosphate site is partially occupied by another sulfate ion that is hydrogen bonded to the side chains of Ser-148 and Thr-208. It also has hydrogen bonds with the main chain of Gly-209 and the side chain of Arg-195 via two water molecules.3 The C3 phosphate of G3P is believed to first bind to the inorganic phosphate site,and then,during the coenzyme exchange step, the acyl group undergoes a conformational isomerization and the C3 phosphate flips to the substrate anion site.3 Arg-195 also contributes to the formation of the anion binding sites.8 Arg-195 is involved with the initial interaction of the C3 phosphate of G3P in the inorganic phosphate site, and may assist with the hemithioacetal intermediate formation.8
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