Cysteine residues glyceraldehyde-3-phosphate dehydrogenase

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

Glyceraldehyde-3-phosphate dehydrogenase has an essential cysteine residue in its active site. The enzyme forms a transient acyl compound with its substrate, glyceraldehyde 3-phosphate, (a) What is the general chemical name of the compound (b) Draw its likely structure. 

Let us consider the mechanism of glyceraldehyde 3-phosphate dehydrogenase in detail (Figure 16.8). In step 1, the aldehyde substrate reacts with the sulfhydryl group of cysteine 149 on the enzyme to form a hemithioacetal. Step 2 is the transfer of a hydride ion to a molecule of NAD + that is tightly bound to the enzyme and is adjacent to the cysteine residue. This reaction is favored by the deprotonation of the hemithioacetal by histidine 176. The products of this reaction are the reduced coenzyme NADH and a thioester intermediate. This thioester intermediate has a free energy close to that of the reactants. In step 3, orthophosphate attacks the thioester to form 1,3-BPG and free the cysteine residue. This displacement occurs only after the NADH formed from the aldehyde oxidation has left the enzyme and been replaced by a second NAD+. The positive charge on the NAD+ may help polarize the thioester intermediate to facilitate the attack by orthophosphate. 

Figure 16.7. Structure of Glyceraldehyde 3-Phosphate Dehydrogenase. The active site includes a cysteine residue and a histidine residue adjacent to a hound NAD+. 

Inactivation of alcohol dehydrogenase from yeast with 14C-labeled [3-(3-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation showed the presence of 1-carboxymethyl histidine66. After inactivation of the enzyme with labeled [3-(4-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation, S-carboxymethyl cysteine was identified in the protein. In the case of glyceraldehyde-3-phosphate dehydrogenase, treatment with either coenzyme analogue leads to the modification of the cysteine residue. Treatment with [14C]nicotinamide-5-bromo-4-methylimidazole dinucleotide did not reveal any modified amino-acid-residues. The labeled nicotinamide residue split off during the recovery of the inactivated enzyme. Attempts to synthesize an inactivator labeled with a 14C-acetyl residue did not give satisfactory yields. If the enzyme-coenzyme derivative was treated with tritiated sodium boron hydride, tritium could be introduced (Fig. 22). Studies with... 

Figure 16.6 Structure of glyceraldehyde 3-phosphate dehydrogenase. Notice that the active site includes a cysteine residue and a histidine residue adjacent to a bound NAD molecule. The sulfur atom of cysteine will link with the substrate to form a transitory thioester intermediate. [Drawn from IGAD.pdb,]...

The molecule of glyceraldehyde-3-phosphate dehydrogenase is a tetramer, consisting of four identical subunits. Each subunit binds one molecule of NAD+, and each subunit contains an essenrial cysteine residue. A thioester involving the cysteine residue is the key intermediate in this reaction. In the phosphoryla-rion step, the thioester acts as a high-energy intermediate (see Ghapter 15 for a... 

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