Phosphofructokinase is like a miniature molecular computer that senses the levels of different molecules and decides if the time is right for breakdown of sugar. Phosphfructokinase is a mechanical computer, with moving parts. The bacterial enzyme PDB entry 4pfk is composed of four identical subunits, but the forms in our own cells are even larger and more complex. Each active site is composed of two different subunits that close around either side of the sugar and ATP molecules.
The whole enzyme shifts when ADP and other molecules bind to regulatory sites between the subunits, shown here with asterisks. Structures have been determined for the active state PDB entry 4pfk and an inactive structure PDB entry 6pfk , revealing that when the enzyme shifts, the shape of the active site is changed and the enzyme switches on and off. A hint: when you are looking at the structures of phosphofructokinase in the PDB, be sure to download the entire biological assembly, which contains four subunits!
Fructose 1,6-bisphosphate aldolase, with a close up of a substrate bound in the active site right. At this stage in glycolysis, the sugar molecule is primed and the cell is ready to start breaking it up. The fourth enzyme, fructose 1,6-bisphosphate aldolase, cuts the molecule in the middle, producing two similar pieces, each with a single phosphate attached.
The enzyme also readily performs the reverse reaction, connecting these two smaller molecules to reform the phosphorylated fructose. In fact, the enzyme is named for this reverse reaction, which is an aldol condensation. The enzyme shown here, from PDB entry 4ald , is found in our muscle cells. It contains four identical subunits, each with its own active site.
The active site uses a special lysine, number in this particular form, to attack the sugar chain. As seen in PDB entry 1j4e , this lysine forms a covalent bond with molecule during the cleavage reaction. This structure is frozen at a stage when the sugar molecule with red oxygen, white carbon, and yellow phosphorus has been cleaved and only half is left in the active site. The aldolase enzyme used by most bacteria is different than the aldolases that we have in our cells. It uses two metal ions instead of a special lysine amino acid.
You can look at an example of a bacterial aldolase in PDB entry 1zen. Be sure to look for the metal ions! Also, take a look at the unusual enzyme made by hot-spring archaebacteria, in PDB entry 1ojx. It uses a lysine in the active site, like our enzyme, but is composed of ten chains in a huge molecular complex. Triose phosphate isomerase, with a close up of a substrate bound in the active site bottom.
Cartoon representation of triose phosphate isomerase, drawn by Jane S. At this stage in glycolysis, the cell has broken the sugar into two different pieces. For economy, it would be ideal to proceed along a single path, instead of requiring a separate path for each of the pieces. The fifth step makes this possible by interconverting the two pieces. Triose phosphate isomerase, shown here from PDB entry 2ypi , pulls a hydrogen atom off one carbon atom and replaces it on a neighboring carbon atom.
A special glutamate amino acid performs the transfer. Triose phosphate isomerase has been described as a perfect enzyme. It performs its reaction billions of times faster than would happen without it. It is so fast that the rate of the reaction is determined by how fast the molecules can get to the enzyme. When you are looking at this structure yourself, also notice that the active site is in the middle of a "beta barrel:" a cylindrical arrangement of extended strands, all colored green in the close-up picture here.
The cartoon picture, drawn by Jane Richardson, shows the folding of the chain in the protein, with an inner ring of extended beta strands surrounded by an outer ring of alpha helices. Notice how each of the helices connects two neighboring strands in the barrel. As you are exploring the structures of the ten glycolytic enzymes, notice that several other enzymes are also constructed with this beautiful folding pattern.
Glyceraldehydephosphate dehydrogenase, with a close-up of the active site right. Halfway through glycolysis, the cell is finally ready to start extracting some energy. In the sixth and seventh steps, the cell will add a new phosphate to each of the molecules, and then use it to make two new ATP molecules. Glyceraldehydephosphate dehydrogenase takes a phosphate ion and connects it to the molecule. In the process, it also extracts two hydrogen atoms using the hydrogen-carrier molecule NAD, colored magenta here.
As mentioned on the first page, these hydrogen atoms may be used to create even more energy using aerobic pathways, or recycled in several ways back onto the broken sugar molecule. Glyceraldehydephosphate dehydrogenase is composed of four identical subunits. Many of the structures of this enzyme in the PDB, such as the human form shown here on the left from PDB entry 3gpd , have NAD bound in all four active sites, as well as two phosphate or sulfate ions.
One ion is bound in the site occupied by the phosphate group in the sugar molecule, and the other is thought to correspond to the site that positions the incoming phosphate ion for the reaction.
PDB entry 1nqo captures the first step in the reaction, when the substrate molecule binds. A nearby cysteine amino acid will then attack the molecule, forming a bond with one carbon atom.
The bond is then broken when the phosphate is attached. In this structure, the cysteine is changed to a less active serine to allow study. A nearby histidine also assists in the reaction. When you are looking at the 1nqo structure, be sure to download the proper biological unit.
The primary PDB file contains four chains, but they are not the proper biological tetramer. Induced fit motion in phosphoglycerate kinase: open form left and closed, reactive form right. Now, at the seventh step of glycolysis, the cell is ready to make some ATP. The glucose has been split into two halves, each of which now has two phosphates attached. Glucosephosphate is rearranged into fructosephosphate such that the molecular formula is unchanged.
Another isomerase is triose phosphate isomerase. It catalyzes the isomerization of dihydroxyacetone phosphate to glyceraldehydephosphate.
The energy released as G3P is oxidized causing subsequent reduction of is highly exergonic. This energy, sometimes referred to as the energy of oxidation, drives the addition of inorganic phosphate onto G3P, yielding the doubly-phosphorylated 1,3-BPG. Hexokinase is the first enzyme in the glycolytic pathway and it is responsible for the phosphorylation of glucose to glucose 6-phosphate.
The other enzymes catalyze subsequent reactions in glycolysis. The rate-limiting step of glycolysis is the conversion of glucosephosphate to fructosephosphate. This reaction is catalyzed by the enzyme phosphofructokinase. Hexokinase catalyzes the conversion of glucose to glucosephosphate, pyruvate kinase converts Phosphoenolpyruvate to pyruvate, and lactate dehydrogenase converts pyruvate into lactose.
Glyceraldehyde 3-phosphate dehydrogenase is the only enzyme in glycolysis that carries out a redox reaction. Glyceraldehyde 3-phosphate is oxidized to 1,3-bisphosphoglycerate while is reduced to. Which of the following choices is responsible for the decarboxylation in the pyruvate dehydrogenase complex? The pyruvate dehydrogenase complex essentially carries out a two part reaction: a decarboxylation and an oxidation. All these choices play important roles in the pyruvate dehydrogenase complex. Thiamine pyrophosphate TPP is the only choice, however, that is responsible for the decarboxylation step.
Lipoamide acts as transporter, transferring the substrate to a distant active site. FAD then reoxidizes lipoamide for the next substrate. CoA is important in producing the substrate. If you've found an issue with this question, please let us know. With the help of the community we can continue to improve our educational resources.
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Example Question 1 : Glycolysis Enzymes. Which of these enzymes catalyzes the first reaction in glycolysis? Possible Answers: Aldolase. Correct answer: Hexokinase. Explanation : The first step in glycolysis is the conversion of glucose to glucosephosphate through the consumption on one ATP molecule. Report an Error. Example Question 2 : Glycolysis Enzymes. Dihydroxyacetone is converted to glyceraldehydephosphate by what category of enzyme?
Possible Answers: Kinase. Correct answer: Isomerase. Possible Answers: glucose. Correct answer: pyruvate. Step 3. The third step is the phosphorylation of fructosephosphate, catalyzed by the enzyme phosphofructokinase. A second ATP molecule donates a high-energy phosphate to fructosephosphate, producing fructose-1,6-bisphosphate. In this pathway, phosphofructokinase is a rate-limiting enzyme. This is a type of end product inhibition, since ATP is the end product of glucose catabolism.
Step 4. The newly added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs an enzyme, aldolase, to cleave 1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone-phosphate and glyceraldehydephosphate. Step 5. In the fifth step, an isomerase transforms the dihydroxyacetone-phosphate into its isomer, glyceraldehydephosphate. Thus, the pathway will continue with two molecules of a single isomer.
At this point in the pathway, there is a net investment of energy from two ATP molecules in the breakdown of one glucose molecule. So far, glycolysis has cost the cell two ATP molecules and produced two small, three-carbon sugar molecules. Both of these molecules will proceed through the second half of the pathway, and sufficient energy will be extracted to pay back the two ATP molecules used as an initial investment and produce a profit for the cell of two additional ATP molecules and two even higher-energy NADH molecules.
Figure 3. Step 6. The sugar is then phosphorylated by the addition of a second phosphate group, producing 1,3-bisphosphoglycerate. Note that the second phosphate group does not require another ATP molecule. Here again is a potential limiting factor for this pathway. If oxygen is available in the system, the NADH will be oxidized readily, though indirectly, and the high-energy electrons from the hydrogen released in this process will be used to produce ATP.
Step 7. In the seventh step, catalyzed by phosphoglycerate kinase an enzyme named for the reverse reaction , 1,3-bisphosphoglycerate donates a high-energy phosphate to ADP, forming one molecule of ATP. This is an example of substrate-level phosphorylation. A carbonyl group on the 1,3-bisphosphoglycerate is oxidized to a carboxyl group, and 3-phosphoglycerate is formed. Step 8.
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