Welcome to part 4 in our glycolysis series. In this section we will focus on the enzymatic activities of glyceroldehyde 3-phosphate dehydrogenase. So we left glycolysis in our last section just after the PFK1 enzyme produced fructose 1-6-bisphosphate. In step 4 of glycolysis, the fructose 1-6-bisphosphate is cleaved by aldolase to yield 1 molecule of dihydroxyacetone phosphate and 1 molecule of glyceroldehyde 3-phosphate. This is followed by the conversion of dihydroxyacetone phosphate to glyceroldehyde 3-phosphate by the action of triose phosphate isomerase. The next reaction step is the first one where we have seen the utilization of an energy carrier molecule to harvest electrons from our food molecules. The glyceroldehyde 3-phosphate dehydrogenase is an oxidoreductase enzyme that oxidizes glyceroldehyde 3-phosphate and reduces the NAD carrier to NADH and a proton. The name Dhydrogenase implies the removal of a hydrogen and its electrons from the molecule which is observed at the C1 position. Also observed is the incorporation of a phosphate group at the C1 position. Thus, the glyceroldehyde 3-phosphate dehydrogenase enzyme mediates two important reactions. In the first, the aldehyde position C1 is oxidized to the carboxylic acid generating the reduced form of the electron carrier NADH. A second dehydration reaction occurs with inorganic phosphate to form the one three-bisphosphoglycerate. Therefore, this enzyme can be classified as two different types of enzymes as the dehydrogenase or oxidoreductase in the first step and as the hydrolyce in the second step. For the purpose of glycolysis, the formation of the NADH is the important component of the reaction from an energy generating standpoint. Thus, the enzyme was named for its ability to mediate this step of the reaction. Here are the two major reaction steps. The oxidation step shown in the upper panel is favored and has a negative delta G value. Therefore, it is spontaneous. However, the dehydration reaction in the second step is not favored and requires an input of energy to go in the forward direction. The enzyme is able to couple these reactions together and use the excess energy from the oxidation reaction to complete the dehydration step in the second part. It does this by forming a thioester covalent intermediate with the enzyme during the reaction mechanism. Let's take a look at this in a little bit more detail. An active site cysteine residue mediates an attack on the glyceraldehyde three-phosphate at the C1 position. The reaction is stabilized by a basic residue in the active site, most likely histidine. The formation of the carbonyl double bond as the electrons from the oxygen flow back into the glyceraldehyde three-phosphate causes the oxidation of the molecule with the loss of the electrons and the hydrogen from the C1 position. The electrons are transferred to the NAD electron carrier molecule forming NADH. NADH leaves the active site of the enzyme and is replaced with another oxidized form of NAD. Inorganic phosphate also enters the active site of the enzyme with the incoming NAD. One of the oxygens from the phosphate mediates attack on the carbonyl carbon forming a covalent bond. The thioel serves as a leaving group, restoring the active site of the enzyme and releasing the product one three-bisphosphoglycerate. In summary, the glyceraldehyde three-phosphate dehydrogenase enzyme mediates the oxidation and dehydration of glyceraldehyde three-phosphate to one three-bisphosphoglycerate. In the process, the enzyme consumes one molecule of inorganic phosphate and produces the reduced form of the NAD cofactor, which is NADH, plus the extra proton that is released by the inorganic phosphate during the dehydration part of the reaction. The NADH carrier will then be utilized under aerobic conditions in the electron transport chain to produce ATP through oxidative phosphorylation. For each glucose molecule, this reaction happens twice, producing two NADH molecules.