The enzyme-substrate complex, which involves weak bonds, is readily reversed. The enzyme-substrate complex formation occurs if the substrate has groups of atoms that are in the correct three-dimensional orientation to interact with the binding atoms in the active site.
Concentrations of substrates, temperature, protons pH of the milieu change the catalytic activity of an enzyme. The catalytic activity of an enzyme is measured by the rate of its reaction proportional to the concentration of the enzyme. It varies according to the substrate concentration through a hyperbolic curve from first order the activity increases approximately linearly with the increase in substrate concentration to zero order the substrate concentration has very little effect on the rate of reaction see FIGURE 3a.
However, the activity of some enzymes shows a sigmoid curve indicating that the activity of that enzyme can be either reduced or activated by small molecules ligands at the same concentration of the substrate see FIGURE 3b. The latter type of enzyme belongs to an "allosteric" model of a better physiological significance.
Indeed, the allosteric enzymes belong to the "substrate cycle system" of reversible reactions where the A to B reaction led by one enzyme is reversed by reaction B to A by another enzyme. The maximal catalytic activity Vmax of an enzyme and its half Vmax maximal velocity , expressed by the Michaelis-Menten constant Km , has a physiological significance which can estimate the metabolic flux of a series of reactions.
The Km value also provides information about the role of similar regulatory enzyme systems in different cells, such as the first step of glucose reaction in the glycolytic pathway glucose to glucosephosphate, G6P FIGURE 3c.
In skeletal muscle, the hexokinase action has a very-low Km, meaning the enzyme is rapidly saturated by its substrate; in liver, the first step by glucokinase is never saturated by higher physiological concentration. Thus, FIGURE 3c demonstrates that muscle glucose uptake is rapidly saturated by normal concentration of arterial glucose, while liver can rapidly phosphorylate this sugar to be converted to glycogen stores after a meal for example.
Another value has a fundamental importance in understanding the relationship between biochemical pathways: the Gibbs free energy concept which combines the first and second laws of thermodynamics. Gibbs, an American scientist, used the definition of enthalpy the heat produced by a reaction together with the entropy molecular disorder induced by the reaction to determine the direction of a reaction of biochemical pathway:.
Practically, the previous enzymatic properties means that one has to take into account the Km of each enzyme involved in the regulatory flux of the pathways, together with the inhibitor and stimulating actors of a precise substrate cycle. This approach is compulsory to understand the several hundred-fold increase of the glycolytic flux as soon as one is facing high intensive exercise. Besides the free energy concept, one has also to consider the coupling of biochemical reactions.
Under those conditions we need to know the precise metabolic flux in Joules or kcal of these irreversible reactions to evaluate the predominance of the real flux between the two reactions J1 and J2.
One example may clarify this concept: the substrate cycle between fructosephosphate F6P and fructose-1,6-bisphosphate F-1,6-BP in muscle glycolysis under resting condition. It may be already visualized that the synthesis to glucose from pyruvate is a much slower process. This observation points out another important fact related to kinetic reaction within a substrate cycle. We may consider the existence of equilibrium and non-equilibrium reactions within a substrate cycle.
When there is a maximum catalytic activity for one enzyme as compared to a much lower activity for the other enzyme, this situation characterises a non-equilibrium process. Conversely, a reaction is near-equilibrium if the activities of the two enzymes are close to each other.
Glycogenolysis and glycolysis within skeletal muscle fibres are regulated by far-from equilibrium enzymes. Glucose taken up by blood supply from liver glycogen is catabolized by 10 enzyme reactions down to pyruvate. Only three substrate cycles induced by far-from equilibrium enzyme systems are involved in the regulation of the glycolytic flux from glucose to pyruvate FIGURE 4. In order to shed light of these regulatory couples, one has to analyze the free energy status of enzymes involved in striated muscle glycolysis 3 TABLE 4.
All three enzymes leading to the production of pyruvate from glucose are far from equilibrium. Moreover, muscle glycogen degradation to glucose molecules is led by another substrate cycle that involves glycogen phosphorylase and glycogen synthase 3 FIGURE 5. Phosphorylation of the two enzymes by ATP and a specific protein kinase has opposite action on glycogen molecule. Doing so during exercise, the release of one glucose molecule is favoured, while glycogen synthesis is inhibited, the two enzymes being next to glycogen molecules.
The entrance of pyruvate into mitochondria is the next step under enzyme regulation. Again, the flux of the CAC is led by three allosteric enzyme systems out of a total of 8 enzymes , all far from equilibrium. However, the limiting flux of the cycle is under the leadership of the lowest activity member, 2-OGDH.
This explain why the oxidative flux of CAC unit of metabolite. From those experimental observations, one has to choose the correct enzyme system to understand why there is no anarchy within a resting muscle. When there is no need to produce new ATP molecules in the oxidative phosphorylation system such at rest , the regulatory enzymes slow down the activation of the glycolytic and mitochondrial pathways, favouring the synthesis of carbohydrates and fat weight while overeating and keeping out of exercise!
The next regulation to investigate, as far as the limitation factor is concerned, is the transfer of free fatty acids into the mitochondria. The fatty acid has to be linked to coenzyme A CoA in the cytoplasm, forming a fatty acid-CoA molecule which is transported across the external membrane of the mitochondria to react with carnitine an amino acid derivative synthesized in the liver and the kidney by a specific enzyme, the fatty acid-carnitine.
The limitation step of fatty acid oxidation is the carnitine acyltransferase complex having a low Km, and thus a rapid saturated flux. It appears that short sprinting lowers PC concentration but has a reduced effect on glycogen stores. On the contrary, long distance endurance running depleted mostly glycogen content but has a moderate depletion in PC store and lactate production. Thus, it seems quite clear that small ligands, such as Pi, ADP leads to the activation of specific regulatory enzymes.
How could we investigate the metabolic adaptation related to any modification of a specific pathway being activated, or not , as evidence of a change in enzyme activity? The answers are: 1 to choose the right limiting enzyme in a metabolic cycle; 2 to measure the quantity of that enzyme. The latter investigation implies the isolation of that enzyme knowing that the metabolic flux is directly related to the amount of enzyme available in that tissue.
For many years, molecular biologists did apply this sequence using highly specific tools. Nevertheless, we now know that mRNA amplifications do not implicitly postulate that new molecules of enzymes proteins have been made up in a tissue.
Moreover, the last decade gave us new tools to investigate biogenesis in skeletal muscle: the discovery of microRNA miRNA that posttranscriptionally regulates the expression of target genes 8. The miRNAs are a class of about 22 nucleotide in all animals, plants and unicellular eukaryotes non-coding RNAs that control diverse biological functions 9.
Over miRNAs have been identified within the human genome, and a single miRNA may inhibit several target genes, thus acting on skeletal muscle differentiation, such as during skeletal myogenesis The increased production of miRNA will slow down the synthesis of new protein molecules by inducing mRNA cleavage, either by translational inhibition and or by promoting the degradation of target mRNA, thus inhibiting the production of new protein molecules.
For certain, miRNAs are playing a major role in explaining adaptations to cardiac and skeletal muscle hypertrophy in resistance exercise training 9 , in sarcopenia Apparently, results from plasma circulating miRNAs might have potential value as physiological mediators of exercise-induced cardiovascular adaptation in athletes Looking to the effects of exercise training, it becomes clear one needs to choose the correct enzymes to evaluate changes of specific metabolic adaptations in skeletal and cardiac muscles.
Numerous publications, even in high standing periodicals, concluded on the consequence of exercise training by inadequate interpretations of correct experimental data using either non-equilibrium enzymes or miRNA modifications without consecutive quantitative and appropriate enzyme results.
As noted by Newsholme and Leech 1 several authors concluded on adaptations induced by exercise training using lactate dehydrogenase LDH , citrate synthase CS , succinate dehydrogenase SDH publications , Other publications use statistical conclusions to postulate a relationship adaptation induced by nutritional supplementation , low-intensity exercise The most stimulating conclusion on exercise adaptations seems to be the comparison between two different animal species: man versus other vertebrates.
TABLE 6 gives us some examples on moving capacities between two species. Comparing the activities of some regulatory enzymes of the carbohydrates and fatty acids substrate of a man and a humming bird, we have to admit the metabolic efficiency of the flying animal is more adapted to release energy for its exercise activities.
The maximal activities of specific enzymes leading to ATP production in the cytosol and mitochondria are from 2 to 3. Eventually, we are still in the indecision state while looking to the precise role of miRNA adaptations induced, or not, by exercise training. Presently, we are aware of the importance of miRNA influence upon adaptations induced by exercise training.
Moreover, a recent publication emphasizes the action of miRNAa upon cardiac ageing and function in humans The quantity of miRNAa is regularly enhanced from childhood to elderly. However, we still need to know details on the precise actions induced through miRNA modifications: what are the signals acting upon some small DNA fragments of miRNA synthesis? Synthesis of miRNAs is reduced to enhance new protein molecules such as during exercise training , while they are increased to slow down the synthesis of enzymes and structure proteins under specific conditions such as ageing.
In March , a paper of Memczak et al. Their data argue that circRNAs can be used as potent inhibitors of miRNAs, thus inducing protein synthesis in specific tissues. For certain it remains to determine if those recent facts could be applied under physical exercise training.
But, once again, what are the signals induced by exercise that stimulate the circRNAs? In order to evaluate the precise biochemical mechanisms involved in exercise conditions, as well as during training practise, it appears compulsory to focus the attention to the regulatory enzymes in the appropriate metabolic pathway.
Enzymes near-equilibrium may be stimulated by general nuclear factors such as several hormones but they will not modify the flux of substrates within a specific pathway. On the contrary, the increase of far-from-equilibrium enzymes are needed to evaluate the real fluxes and adaptations observed as a consequence of metabolic increase, especially in substrate transport, the Krebs cycle, and oxidative phosphorylation.
Those regulations seem to be under the expression of circRNAs and mi-RNAs which either increase or reduce protein molecule synthesis. We still need to identify the factors acting on circRNAs and miRNAs synthesis under exercise condition and training adaptation.
Open menu Brazil. About the journal Editorial Board Instructions to authors Contact. Open menu. Text EN Text English. Training; Enzymes; Non-equilibrium reactions; Regulatory pathways. Key words: Training; Enzymes; Non-equilibrium reactions; Regulatory pathways.
This interaction includes the enzymatic control of each pathway, each organ's metabolic profile and hormone control. Flow is regulated in the gluconeogenesis-specific reactions. Pyruvate carboxilase is activated by acetyl-CoA, which signals the abundance of citric acid cycle intermediates, i. The citric acid cycle is regulated mostly by substrate availability, product inhibition and by some cycle intermediates.
Carbamoyl-phosphate sinthetase is stimulated by N-acetylglutamine, which signals the presence of high amounts of nitrogen in the body. Liver contains a hexokinase hexokinase D or glucokinase with low affinity for glucose which unlike "regular" hexokinase is not subject to product inhibition. Therefore, glucose is only phosphrylated in the liver when it is present in very high concentrations i. In this way, the liver will not compete with other tissues for glucose when this sugar is scarce, but will accumulate high levels of glucose for glycogen synthesis right after a meal.
Acyl-CoA movement into the mitochondrion is a crucial factor in regulation. Malonyl-CoA which is present in the cytoplasm in high amounts when metabolic fuels are abundant inhibits carnitine acyltransferase, thereby preventing acyl-CoA from entering the mitochondrion. Furthermore, 3-hydroxyacyl-CoA dehydrogenase is inhibited by NADH and thiolase is inhibited by acetyl-CoA, so that fatty acids wil not be oxidized when there are plenty of energy-yielding substrates in the cell. Usually neurons use only glucose as energy source.
Since the brain stores only a very small amount of glycogen, it needs a steady supply of glucose. During long fasts, it becomes able to oxidize ketone bodies. The maintenance of a fairly steady concentration of glucose in the blood is one of the liver's main functions. This is accomplished through gluconeogenesis and glycogen synthesis and degradation. It synthesizes ketone bodies when acetyl-CoA is plenty. It is also the site of urea synthesis.
It synthesizes fatty acids and stores them as triacylglycerols. Glucagon activates a hormone-sensitive lipase, which hydrolizes triacylglycerols yielding glycerol and fatty acids. These are then released into the bloodstream in lipoproteins. Muscles use glucose, fatty acids, ketone bodies and aminoacids as energy source.
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