Glycolysis (enzymes of the payoff phase)

The payoff phase, as I mentioned earlier, concerns the whole of the last five reactions of glycolysis and allows the cell to obtain energy in this process. Here are some ideas on the enzymes of phase 5 payoff ...
6^th enzyme – Glyceraldehyde-3-phosphate dehydrogenase

This enzyme, often abbreviated to GAPDH, is presented in the form of a tetramer. Each subunit has about 35.9 kDa (331 amino acids) and shows how a molecule of cofactor NAD+. The subunits are designated by O, P, Q and R and are independent of each other. That is, each subunit catalyses the reaction without the intervention of others. As described in the post about the reactions of the payoff phase, the reaction catalyzed by this enzyme is a double one, involving an oxidation and an addition of a phosphate group. It is an enzyme that may be affected by the presence of arsenic in the body, causing the yield of glycolysis to become null. Its mechanism of action involves both a covalent catalysis and acid-base. To do this, it is essential the participation of cysteine ​​149 and histidine 176 for both types of catalysis, resectivamente. The substrate binds covalently to cysteine​​, forming a hemitioacetal. The laboratory level this enzyme is widely used (I also use ...) as a positive control techniques such as immunoblotting or RT-PCR, because in general their expression is constant in almost all cell types. So it is possible to determine changes in the expression of a certain gene or in the presence of a given protein by comparing it with the levels of GAPDH.

7^th enzyme – Phosphoglycerate kinase

This enzyme requires Mg²⁺ to make its catalytic activity. The name derives from the reaction of the enzyme in the reverse direction, which occurs during photosynthetic CO₂ fixation. It is responsible for the production of the first molecules of ATP in glycolysis. Its amino acid sequence has to be extremely conserved in different organisms. The monomeric enzyme is composed of two domains of equivalent size, which corresponds to half N-and C-terminal. The substrate (1,3-bisphosphoglycerate) binds to the first half, while ADP binds to the second. Presents a sequential kinetic mechanism in which catalysis occurs by a proximity effect.
8^th enzyme 8th – Phosphoglycerate mutase

The phosphoglycerate mutase is dimeric, with each of its subunits with about 32kDa. As the name implies, this is a mutase enzyme, ie, catalyzes the transfer of phosphoryl groups within a molecule. In other words, it changes the position of phosphoryl groups. In fact, the enzyme is phosphorylated (fosfoenzima is one), and will give up its phosphoryl group to the carbon of the substrate 2, resulting in an intermediate with two phosphoryl groups (2,3-bisphosphoglycerate). Only after this step, is that the phosphoryl group that was originally in the substrate (position 3) is removed, regenerating the initial form (phosphorylated) enzyme.

The phosphoglycerate mutase has three different isoforms (isozymes or isoenzymes), predominantly found in cardiac muscle, skeletal muscle and the third one in the other tissues.

9th enzyme – Enolase

The enolase is a dimeric metalloenzyme, and each subunit has about 40-50 kDa. These subunits have an antiparallel orientation, interacting with each other via two salt bridges, involving an arginine and a glutamate each. The N-terminal domain of alpha-3 subunit has four helices and beta sheets. The C-terminal domain has two beta sheets and two alpha-helices, and it ends with a barrel consists of beta sheets and alpha helices alternate. The two Mg²⁺ ions required for catalytic activity are critical in neutralizing negative charges. This enzyme has a pH optimum of about 6.5, and can also be called fosfopiruvato dehydratase. It was initially discovered in 1934 by researchers Lohmann and Meyerhof. As with the enzyme before the enolase also has three different isoforms, of which one is predominantly found in muscle tissue, the other in neurons and the third one in the remaining parts of the body.
The enolase is inhibited by fluoride ion, and this fact is exploited, for example, when collecting blood samples for analysis. In this case, when it is important to inhibit glycolysis (to keep unchanged the concentration of serum glucose), blood can be collected in tubes containing fluoride.
10^th enzyme – Pyruvate kinase

This enzyme is responsible for the second ATP production in glycolysis and is the third regulatory enzyme of this pathway. It needs the presence of two metal ions: K⁺ and Mg²⁺ (or Mn²⁺). It has four different isoforms, one located predominantly in the liver, another in red blood cells, the other in cardiac and skeletal muscle and brain and the latter is mainly found in fetal tissues. It is a tetrameric enzyme, each subunit has about 500 amino acids.

Main bibliographic sources:

- Voet D, Voet JG, Biochemistry, Wiley

- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers