datomic bomb

There are four levels of protein structure. The primary structure refers to the sequence of amino acid residues in the polypeptide chain written left-to-right from the N-terminus to the C-terminus. Secondary structures are ordered structures formed by internal hydrogen bonding between amino acid residues. The common secondary structures are the α helix, the β strand, and various loops and turns. The β sheet is often counted as secondary structure although, strictly speaking, it is a motif.

(--> The next level of protein structure, secondary structure, refers to local folded structures that form within a polypeptide due to interactions between atoms of the backbone. (The backbone just refers to the polypeptide chain apart from the R groups – so all we mean here is that secondary structure does not involve R group atoms.) The most common types of secondary structures are the α helix and the β pleated sheet. Both structures are held in shape by hydrogen bonds, which form between the carbonyl O of one amino acid and the amino H of another. - Khan A.)

The tertiary structure of a polypeptide is the three-dimensional conformation. Typical proteins contain α helices, β strands, and turns, although there are some proteins that only have α helices and turns, and others that have only β sheets and turns. In many cases, the final structure consists of distinct, independently  folded regions called domains. An example of a protein with multiple domains is shown on the left. This protein is the enzyme pyruvate kinase from cat (Felix domesticus). There are three separate domains indicated by the square brackets on the side. Note that each of the domains is connected to another by a short stretch of unordered polypeptide chain. In some cases, a particular domain is shared by several proteins suggesting that different proteins can be formed by combining various domains that evolved separately. In other cases, similar domain structures might arise independently by convergent evolution.

(--> The overall three-dimensional structure of a polypeptide is called its tertiary structure. The tertiary structure is primarily due to interactions between the R groups of the amino acids that make up the protein. R group interactions that contribute to tertiary structure  include hydrogen bonding, ionic bonding, dipole-dipole interactions, and London dispersion forces – basically, the whole gamut of non-covalent bonds. For example, R groups with like charges repel one another, while those with opposite charges can form an ionic bond. Similarly, polar R groups can form hydrogen bonds and other  dipole-dipole interactions. Also important to tertiary structure are hydrophobic interactions, in which amino acids with nonpolar, hydrophobic R groups cluster together on the inside of the protein, leaving hydrophilic amino acids on the outside to interact with surrounding water molecules. Finally, there’s one special type of covalent bond that can contribute to tertiary structure: the disulfide bond. Disulfide bonds, covalent linkages between the sulfur-containing side chains of cysteines, are much stronger than the other types of bonds that contribute to tertiary structure. They act like molecular "safety pins," keeping parts of the polypeptide firmly attached to one another. - Khan A.)

Quaternary structure only applies to proteins that are composed of more than one polypeptide chain. Each of the polypeptides is called a subunit. The subunits might be identical, as in the example shown above, or they might be very different as in my favorite enzyme ubiquinone:cytochrome c oxidoreductase (complex III).

(--> Many proteins are made up of a single polypeptide chain and have only three levels of structure (the ones we’ve just discussed). However, some proteins are made up of multiple polypeptide chains, also known as subunits. When these subunits come together, they give the protein its quaternary structure.We’ve already encountered one example of a protein with quaternary structure: hemoglobin. As mentioned earlier, hemoglobin carries oxygen in the blood and is made up of four subunits, two each of the α and β types. Another example is DNA polymerase, an  enzyme that synthesizes new strands of DNA and is composed of ten subunits5^5​5​​start superscript, 5, end superscript.In general, the same types of interactions that contribute to tertiary structure (mostly weak interactions, such as hydrogen bonding and London dispersion forces) also hold the subunits together to give quaternary structure. - Khan A.)

There are certain motifs that occur over and over again in different proteins. The helix-loop-helix motif, for example, consists of two α helices joined by a reverse turn. The Greek key motif consists of four antiparallel β strands in a β sheet where the order of the strands along the polypeptide chain is 4, 1, 2, 3. The β sandwich is two layers of β sheet [see β Strands and β Sheets].

The vast majority of motifs do not have a common evolutionary origin in spite of many claims to the contrary. They arise independently and converge on a common stable structure. The fact that these same motifs occur in hundreds of different proteins indicates that there are a limited number of possible folds in the universe of protein structures. The original primitive protein may have been relatively unstructured but over time there will be selection for more and more stable structures. This selection will favor the common motifs.

Larger motifs are often called domain folds because they make up the core of a domain. The parallel twisted sheet is found in many domains that have no obvious relationship other than the fact that they share this very stable core structure. The β barrel structure is found in many membrane proteins. There are dozens of enzymes that have adapted to an α/β barrel. These enzymes are not evolutionarily related. (The β helix is much less common.)

Denaturation and protein folding

Each protein has its own unique shape. If the temperature or pH of a protein's environment is changed, or if it is exposed to chemicals, these interactions may be disrupted, causing the protein to lose its three-dimensional structure and turn back into an unstructured string of amino acids. When a protein loses its higher-order structure, but not its primary sequence, it is said to be denatured.

Denatured proteins are usually non-functional.For some proteins, denaturation can be reversed. Since the primary structure of the polypeptide is still intact (the amino acids haven’t split up), it may be able to re-fold into its functional form if it's returned to its normal environment. Other times, however,  denaturation is permanent. One example of irreversible protein denaturation is when an egg is fried. The albumin protein in the liquid egg white becomes opaque and solid as it is denatured by the heat of the stove, and will not return to its original, raw-egg state even when cooled down.Researchers have found that some proteins can re-fold after denaturation even when they are  alone in a test tube. Since these proteins can go from unstructured to folded all by themselves, their amino acid sequences must contain all the information needed for folding. However, not all proteins are able to pull off this trick, and how proteins normally fold in a cell appears to be more complicated. Many proteins don’t fold by themselves, but instead get assistance from  chaperone proteins (chaperonins).

Glycolysis provides different cell molecules important for cellular proper functioning. The main donor of chemical energy in most of our processes is ATP. However, it also provides precursors for many other processes such as synthesis of amino acids, for example. Thus, it is essential to have a strict regulation of glycolysis, so that the cell can respond to different needs of ATP or other metabolites. During his studies on fermentation of glucose by yeast, Louis Pasteur discovered that the rate and amount of glucose consumed was higher in anaerobic than in aerobic conditions! At first glance this may seem strange, because aerobic metabolism is normally associated with something more advantageous for the cell. In fact, the biochemical explanation is simple: under anaerobic conditions one molecule of glucose generates 2 molecules of ATP, but under aerobic conditions generates 30 or 32 molecules of ATP. Simplifying, if one thinks that in five minutes the yeast will need to get 30 ATP, this means that under aerobic conditions it will only need to spend a molecule of glucose in that time, while under anaerobic conditions, as the process is less profitable in the energy point of view, it is necessary to spend 15 molecules of glucose. That is, the flow of glucose through the glycolytic pathway is regulated depending on cell ATP levels (as well as adequate supplies of glycolytic intermediates to biosynthetic roles).

Glycolysis has 10 reactions, and there are three regulatory points (irreversible reactions). The enzymes that catalyze them are the hexokinase , PFK-1 )and pyruvate kinase . As these reactions are the limiting steps of glycolysis, changes in speed of action of their enzymes will alter the overall speed of the glycolytic pathway. Of the three regulatory enzymes, the main one is PFK-1. This may seem strange, because indeed the most logical situation was that the main regulatory enzyme was the first ... Again, there is a very simple explanation for this. What is happening is that hexokinase is an enzyme also common to other metabolic processes (synthesis of glycogen and pentose phosphate pathway). In other words, despite being a regulatory enzyme, it is not unique to glycolysis. Thus, the main point of regulation of glycolysis has to be the second, ie, the reaction catalyzed by PFK-1.

Activators of hexokinase:

- Fructose-1-phosphate (liver) – It competes with fructose-6-phosphate to the regulatory protein of glucocinase, canceling its inhibitory effect.

- Inorganic phosphate (Pi) – It is a player in the glycolytic process (involved in reaction 6) so it makes sense that if it has a regulatory role, is a stimulating one.

Inhibitors of hexokinase:

- Glucose-6-phosphate (muscle) - It makes sense that functions as an inhibitor because it is the reaction product. If we have too much product, we will not need to continue to produce more.

- Fructose-6-phosphate (liver) – It is the product of the following reaction (reaction 2), but can be interpreted the same way as the molecule before. That is, if we are to accumulate the intermediate formed from the reaction product, there is no point in continuing to make more product. This inhibition occurs through a protein called regulator protein of glucocinase.

Activators of PFK-1 (Phosphofructokinase-1):

- Fructose-2,6-bisphosphate (liver) – It is the most significant allosteric regulator of PFK-1, reducing its affinity for the inhibitors ATP and citrate. It is produced in response to insulin and degraded in response to glucagon.

- Fructose-6-phosphate – It is the substrate, so it makes sense that if we have much substrate the enzyme is activated.

- ADP and AMP – They are produced when ATP is spent, thus indicating a low energy state. Therefore, it makes perfect sense that they can activate glycolysis, so that the cell can replenish their normal energy values. They activate the enzyme because they relieve the inhibition caused by ATP.

Inhibitors of PFK-1:

- Glucagon (liver) - This hormone is produced in a state of hypoglycemia and aims to raise the concentration of glucose in the blood. So it makes perfect sense that it inhibits glycolysis, because this process consumes glucose, which will further accentuate the reduced blood glucose concentration. As mentioned earlier, the glucagon decreases the levels of fructose-2,6-bisphosphate

- ATP - The main objective of glycolysis is to produce energy (ATP). So if the cell already has ATP, it makes sense that glycolysis is inhibited, thus preventing an unnecessary waste of a precious metabolic fuel as glucose! ATP inhibits PFK-1 because it decreases the affinity of the enzyme for its substrate, fructose-6-phosphate.

- Citrate – It stresses the inhibitory effect of ATP. This molecule is the first intermediate of the following step of aerobic catabolism, the Krebs cycle. So if we are accumulating Krebs cycle intermediates, it is useless to continue to perform glycolysis.

- Phosphoenolpyruvate – It is an intermediate of glycolysis that is formed in the penultimate reaction. If there is an accumulation of this intermediate, the reactions above have to be inhibited in order to prevent a further accumulation of the molecule.

- H

+

- This enzyme is particularly sensitive to changes in pH, functioning as a "switch" that turns off, for example, when we make an exaggerated lactic fermentation (produces H

+

), preventing an even greater acidification.

Activators of pyruvate kinase:

- ADP - The reason is the same as mentioned above for the PFK-1, ie, is an indicator of an energy deficit, so it will lead to an activation of glycolysis.

- Fructose 1,6-bisphosphate - an intermediate of glycolysis that is formed in a reaction prior to the one catalyzed by pyruvate kinase. So if we are accumulating an intermediate produced in an earlier stage, we have to activate this enzyme in order to counteract this accumulation (as when a dam is accumulating too much water, and to restore normal values ​​is necessary to open the gate ...).

- Dephosphorylation (liver) - Induced, for example, by insulin, which makes sense, given that insulin is produced in a situation of excess blood sugar (hyperglycemia) and will activate the process (one of them is glycolysis!) that consume glucose in order to lower the blood glucose concentration.

Inhibitors of pyruvate kinase:

- ATP – It is a carrier of chemical energy and one of the end products of glycolysis, so there is no need to continue the breakdown of glucose. It decreases the affinity of the enzyme for phosphoenolpyruvate.

- Acetyl-CoA – It is the molecule in which the product of this reaction (pyruvate) is converted in the case of aerobic catabolism. Therefore, if acetyl-CoA accumulates, it makes no sense to continue to synthesize pyruvate, so the enzyme is inhibited.

- Long-chain fatty acids.

- Phosphorylation (liver) - Induced, for example, by the action of glucagon, which, as mentioned earlier, will have as main function to raise blood glucose levels. To this end, it inhibits, for example, glycolysis.

- NADH - as we shall see in more detail when I speak of cellular respiration, NADH has potential to create molecules of ATP, which signals a high energy state of the cell. In this situation, it is not necessary to resort to glycolysis for more energy.

- Alanine - This amino acid (one of the 20 standard amino acids) can lead to pyruvate (the reaction product of pyruvate kinase!), by removal of its amino group. So if there is a molecule that can directly lead to pyruvate, we do not need to spend more glucose.

In short, we can make some generalizations about the regulation of metabolic pathways, which will be useful to understand the regulation of other processes. First, energy molecules such as ATP, or potential energy, such as NADH are, in general, inhibitors of catabolism. This is very easy to understand if we think that the main objective of the catabolism is to produce energy. If the cell already has this ability it does not need to degrade more nutrients to produce energy! The oppposite reasoning applies to ADP, AMP and NAD+, because any one of these molecules indicates an energy deficit in the cell (remember that when we spend ATP we obtain ADP or AMP, and when we spend NADH we obtain NAD+...) so it will be necessary to restore energy levels, and this activates the catabolism. Second, the reaction product or intermediates formed from this (products of reactions following the reaction we are considering) are inhibitors. On the other hand, the substrate, or intermediaries which will originate the substrate (formed in reactions prior to the reaction that we are considering) are activators.

Citrate synthase The citrate synthase is an enzyme widely used as a biomarker for the presence of intact mitochondria in cell cultures or organelle preparations. Despite being a mitochondrial enzyme it is encoded by nuclear DNA and synthesized in the cytosol. This enzyme is the first regulatory enzyme in the Krebs cycle. It uses two different substrates, the acetyl-CoA and oxaloacetate. The oxaloacetate firstly binds to the enzyme, which induces conformational changes that create the binding site for the acetyl-CoA molecule.

From a structural point of view, it is composed of 437 amino acid residues and has two subunits, each with about 20 alpha helices. The active center has three amino acid residues essential for the catalytic function of the enzyme, due to the establishment of specific interactions with the substrates - His274, His320, and Asp-375.

The aconitase is an enzyme that has a functional iron-sulfur cluster [Fe4S4]2+, which interacts with three cysteine ​​residues of the enzyme. It is especially sensitive to oxidative stress and, in particular, to superoxide anion, due to the iron-sulfur cluster. It has two homologues in our body, the iron-responsive element-binding protein (IRE-BP) and the 2-isopropylmalate dehydratase (or alpha-isopropylmalate isomerase). From a structural standpoint, the aconitase has two conformations, one for the inactive and one for the active state. In the inactive form, it has four domains, the first three establish interactions with the iron-sulfur cluster, while the latter has the active center. When it becomes active, the enzyme is altered in the iron-sulfur cluster (Fe3S4 turns in Fe4S4), and this represents the main difference between the two conformations of the enzyme.

Its mechanism of action relies on a mechanism of dehydration-hydration, via the intermediate cis-aconitate.

Its active site has two amino acid residues particularly important for catalytic activity - His101 and Ser642. The importance of this enzyme, in a physiological point of view, is supported by the existence of many diseases that affect it. One is referred to as aconitase deficiency. It is caused by a mutation in the gene that codes for a protein responsible for the assembling of the iron-sulfur cluster. This disease causes myopathy and exercise intolerance, because the aerobic catabolism of these individuals is compromised. Another disease is Friedreich's ataxia (FRDA), characterized by a lower activity of aconitase and other Krebs cycle enzyme, the succinate dehydrogenase. Besides these, there are studies that suggest a possible relationship between aconitase and diabetes. However, it is still an hypothesis that has to be best characterized.

Isocitrate dehydrogenase 

Isocitrate dehydrogenase is the second regulatory enzyme in the Krebs cycle. There are three different isoforms of isocitrate dehydrogenase. One exists only in the mitochondrial matrix and uses NAD+ as the acceptor of electrons. The other isoforms use NADP+ as the acceptor of electrons and appear to have as main function the formation of NADPH, essential for the reducing anabolic reactions. These forms are present in the mitochondrial matrix, the cytosol and in the peroxisome. The forms using NADP+ as a cofactor have an homodimeric structure, while the one that uses NAD+ is a heterotetramer.

The reaction catalyzed by isocitrate dehydrogenase involves the formation of an intermediary, the oxalossuccinate. From the clinical point of view, some mutations were found in isocitrate dehydrogenase in some brain tumors, including astrocytoma, oligodendroglioma and multiforme glioblastoma. There are also some studies that indicate a possible relationship between mutations in the enzyme and acute myeloid leukemia.

Alpha-ketoglutarate dehydrogenase