C7. Inhibition by Temperature and pH Changes - Biology

C7. Inhibition by Temperature  and pH Changes - Biology

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From 0 to about 40-50° C, enzyme activity usually increases, as do the rates of most reactions in the absence of catalysts. At higher temperatures, the activity decreases dramatically as the enzyme denatures.

Figure: Temperature and Enzyme Activity


pH has a marked effect on the velocity of enzyme-catalyzed reactions.

Figure: pH and Enzyme Activity

Think of all the things that pH changes might affect. It might

  • affect E in ways to alter the binding of S to E, which would affect Km
  • affect E in ways to alter the actual catalysis of bound S, which would affect kcat
  • affect E by globally changing the conformation of the protein
  • affect S by altering the protonation state of the substrate

The easiest assumption is that certain side chains necessary for catalysis must be in the correct protonation state. Thus, some side chain, with an apparent pKa of around 6, must be deprotonated for optimal activity of trypsin which shows an increase in activity with the increase centered at pH 6. Which amino acid side chain would be a likely candidate?

See the following figure which shows how pH effects on enzyme kinetics can be modeled at the chemical and mathematical level.

Figure: Chemical equations showing the mechanism of pH effects on enzyme catalyzed reactions

Figure: Mathematic equations modeling pH effects on enzyme catalyzed reactions

Figure: Graphs of pH effects on enzyme catalyzed reactions

The aim of this study was to determine the effects of seasonal temperature variation on the functional properties of lactate dehydrogenase (LDH) from white muscle and liver of Norwegian coastal cod (Gadus morhua) and the possible relevance of LDH allelic variability for thermal acclimation. Two groups of fishes were acclimated to 4°C or 12°C for one year. Polymorphism was observed in only one (Ldh-B) of the three Ldh loci expressed in cod liver and/or muscle. Isozyme expression remained unchanged regardless of acclimation temperature(TA). The products of locus Ldh-B comprise only 14–19% (depending on the tissue) of total LDH activities and,consequently, differences between phenotypes are negligible in terms of their effect on LDH total performance. No kinetic( ⁠

In conclusion, the strategies of LDH adjustment to seasonal temperature variations in cod involve changes in LDH concentration (quantitative),adjustment of thermodynamic (Ea) and kinetic( ⁠

Factors Affecting Enzymatic Speed

1. Substrate concentration
2. Temperature & pH *
3. Enzyme concentration

Enzymes can be denatured - they change shape so much that they are no longer effective. High temp or pH can cause denaturation.

Enzymatic Inhibition -

Competitive Inhibition and Noncompetitive Inhibition

**Both are forms of feedback inhibition

Some inhibitors are NOT reversible - poisons like cyanide, lead poisoning all affect enzymes

QUESTION: What type of inhibition is pictured below?

Effects of Temperature, Ph, Enzyme Concentration, and Substrate Concentration on Enzymatic Activity

Effects of Temperature, pH, Enzyme Concentration, and Substrate Concentration on Enzymatic Activity INTRODUCTION
Enzymes, proteins that act as catalysts, are the most important type of protein[1]. Catalysts speed up chemical reactions and can go without being used up or changed [3] Without enzymes, the biochemical reactions that take place will react too slowly to keep up with the metabolic needs and the life functions of organisms. Catecholase is a reaction between oxygen and catechol [2]. In the presence of oxygen, the removal of two hydrogen atoms oxidizes the compound catechol, as a result of the formation of water [2]. Oxygen is reduced by the addition of two hydrogen atoms, which also forms water, after catechol is converted to benzoquinone [2]. Long branched chains, the structural backbones of the red and brown melanoid pigments that cause darkening, are formed when the benzoquinone molecules are linked together [2].

Enzymes have a three-dimensional structure that is very complex [2]. This three-dimensional structure consists of one or more polypeptide chains. These polypeptide chains form an active site, an area into which the substrate will fit.

There are four factors that will have an effect on the structure of an enzyme’s active site, the activity of the enzyme, and the rate of the reaction in which the enzyme is involved. The four factors that can affect the activity of an enzyme include temperature, pH, enzyme concentration, and substrate concentration.

In the effects of temperature on enzyme activity, the rate of an enzyme-catalyzed reaction increases at temperature increases, up to the point at which the rate is its maximum [2]. Most enzymes active in living tissue becomes denatured, their secondary or tertiary protein structure breaks down, at the temperature above 40°C [2].

In the effects of pH on enzyme activity, the way a protein folds can be changed in the presence of various ions that can interfere with the pattern of positive and negative charges within a protein molecule [2]. In result, the shapes of the enzyme’s active site may be changed. It is expected that the changes in pH would have an effect on the action of enzymes. In this case, optimum pH is the most favorable pH value because it is the point at which the enzyme is most active [2]. Not only can denaturation be brought on by extremes in temperature, extremely high of low pH values can result in a complete loss of enzyme activity [2].

In the effects of enzyme concentration, an enzyme-substrate complex is formed when a substrate fits into the active of an enzyme [2]. The reaction rate that the enzyme has with the chemical reaction is usually directly proportional to the enzyme concentration [2]. Then, a product is formed. If the substrate is present in excess amounts, the reaction rate will increase in proportion to an increasing enzyme concentration, so that the available substrate does not limit the rate of reaction [2].

In the effects of substrate concentration on enzyme concentration, the velocity, the rate of speed, at which the enzyme works will increase until it reaches a maximum [2]. This is only possible if the amount of enzyme is kept constant but the amount of substrate is gradually increased. Because of the entire available enzyme that is participating in the enzyme substrate complex, there will be an increase in substrate concentration, but it will not increase the velocity of the reaction [2].

In order to investigate the effects of enzymatic activity an experiment will take place to see how changes in the temperature, pH, enzyme concentration, and substrate concentration will cause this effect.

The hypothesis is as the temperature increases, above 40° C, the activity of the enzyme catalyst will increase. The hypothesis for the enzyme activity with the effect of the pH is if the pH is not at the optimum pH value there will.

References: CITED
[1]LCampbell, Neil., Jane Reece.2005. Biology, 7th ed. Beth Wilbur. Benjamin Cummings Publishing Menlo Park, California. pp. 150-157.
[2]Helms, Doris., Carl Helms., Robert Kosinski., John Cummings. 1998. Biology in the Laboratory, 3rd ed, Judith Wilson ed. Freeman Publishing, New York, New York. pp. 10-1 – 10-18.
[3]Timberlake, Karen C.2002. Chemistry: Structures of Life. Ben Robert. Benjamin Cummings Publishing, Menlo Park, California. pp. 208.

Blood Glucose

The amount of glucose in your blood is carefully controlled. Again, this uses the hormonal system. The hormones responsible for regulating blood glucose are produced in the pancreas in particular areas called islets of Langerhans.

After you have eaten a meal, the blood glucose levels will begin to rise because the carbohydrates in the food are digested and absorbed. This rise is detected by beta cells, which then will produce more insulin. This insulin then binds to receptor proteins in cell membranes (particularly in the liver). This causes more protein channels to open so that more glucose can enter the cell. As well as this, insulin encourages enzymes to convert glucose to glycogen (glycogenesis) for storage.

If however, you have been doing a lot of exercises, and glucose is being used up, then alpha cells will produce glucagon, this causes the release of an enzyme that breaks glycogen to glucose (glycogenolysis [gli-ko-jen-oh-li-sis]).

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Fogg, G. E. (1981). The ecology of an extracellular metabolite of seaweeds. In: Fogg, G. E., Jones, W. E. (ed.) Proc. 8. Int. Seaweed Symp. United College of North Wales, Menai Bridge, pp. 46–53

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Norkrans ( 1966) reported that Saccharomyces cerevisiae stops growing at pH 8.0. More recently, the effect of high pH (usually 8.0) has been studied regarding changes of gene expression and synthesis of several enzymes (Vogel and Hinnen 1990 Peñalva and Arst 2002 Platara et al., 2006 Ariño 2010), but their role in the response of this yeast to high pH in terms of significant physiological or biochemical changes is not clear.

Rothstein and Demis ( 1953) showed that both the addition of K + at low pH but also increasing the pH of the medium to around 6.0 increased yeast fermentation. K + and high pH in an exclusive way of one another could not only stimulate fermentation, but also respiration (Peña et al., 1969). This is due to the simulation of the H + -ATPase activity of the plasma membrane (Peña et al., 1972), which is the basic mechanism for acidification and K + transport in yeast.

It is known that, depending on the incubation pH, S. cerevisae shows large changes of the internal pH (Conway and Downey 1950 Peña et al., 1972, 1995 Calahorra et al., 1998). However, it is also sustained that particularly S. cerevisiae maintains its internal pH within narrow limits (Hong et al., 1999 Yenush et al., 2005). Studies have been carried out on the effects of variable pH, usually below 6.0 however, except for some experiments performed at pH 7.5 (Peña et al., 1972), little is known about the metabolic and physiological changes produced at high or extreme values preventing yeast growth. This study represents an attempt to define whether or not yeast cells (S. cerevisiae) stop growing at high pH values due to the inhibition of these general processes might be responsible for growth to stop. The main ones studied after incubating the cells at high pH were first fermentation and respiration as the main sources of energy necessary for other functions: (a) the capacity of the cells to acidify the medium, (b) the ability to generate a plasma membrane potential (PMP) difference, (c) their capacity to transport ions inside and (d) their ability to transport and to incorporate amino acids. Having not found an inhibition, but the stimulation of some of them, our studies carried out on the changes of the cell cycle, that showed its arrest, a microarray analysis of several genes that might be implied in such arrest were performed, revealing changes involved in this phenomenon.

Problem: Which of the following is true of enzymes?a. Enzyme function is independent of physical and chemical environmental factors such as pH and temperatureb. Nonprotein cofactors alter the substrate specificity of enzymesc. Enzymes increase the rate of chemical reaction by lowering activation energy barriersd. Enzyme function is often increased if the 3-D structure or conformation of an enzyme is alterede. Enzymes increase the rate of chemical reaction by providing activation energy to the substrateYou have an enzymatic reaction proceeding at the optimum pH and optimum temperature. You add a competitive inhibitor to the reaction and notice that the reaction slows down. What can you do to speed the reaction up again?a. Increase the pH.b. Add more substrate it will outcompete the inhibitor and increase the reaction rate.c. Increase the temperatured. Add more inhibitor to speed up the reaction.

a. Enzyme function is independent of physical and chemical environmental factors such as pH and temperature
b. Nonprotein cofactors alter the substrate specificity of enzymes
c. Enzymes increase the rate of chemical reaction by lowering activation energy barriers
d. Enzyme function is often increased if the 3-D structure or conformation of an enzyme is altered
e. Enzymes increase the rate of chemical reaction by providing activation energy to the substrate

You have an enzymatic reaction proceeding at the optimum pH and optimum temperature. You add a competitive inhibitor to the reaction and notice that the reaction slows down. What can you do to speed the reaction up again?

a. Increase the pH.
b. Add more substrate it will outcompete the inhibitor and increase the reaction rate.
c. Increase the temperature
d. Add more inhibitor to speed up the reaction.

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Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition.

Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory.

Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can be used to explain why inhibition occurs.

The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favorable. If we consider the enzyme as the lock and the substrate the key (Figure 9) - the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally.

Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. Figure 10.

Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11 shows the reaction velocity decreasing after the maximum velocity has been reached.

Additional amounts of substrate added to the reaction mixture after this point actually decrease the reaction rate. This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 12) and prevent any other substrate molecules from occupying them.

This causes the reaction rate to drop since all of the enzyme present is not being used.

Enzyme Inhibition

In addition to temperature and pH changes, other factors can result in an enzyme's activity being diminished or shut down. In a process called an allosteric interaction, the shape of the enzyme is temporarily changed when a molecule binds to a portion of it away from where it joins the reactant. This leads to a loss of function. Sometimes this is useful when the product itself serves as the allosteric inhibitor, because this is usually a sign of the reaction having proceeded to the point where additional product is no longer required.

In competitive inhibition, a substance called a regulatory compound competes with the reactant for the binding site. This is akin to trying to put several working keys into the same lock at the same time. If enough of these regulatory compounds join to a high enough amount of the enzyme present, it slows or shuts down the reaction pathway. This can be helpful in pharmacology because microbiologists can design compounds that compete with the binding sites of bacterial enzymes, making it much harder for the bacteria to cause disease or survive in the human body, period.

In noncompetitive inhibition, an inhibitory molecule binds to the enzyme at a spot different from the active site, similar to what happens in an allosteric interaction. Irreversible inhibition occurs when the inhibitor permanently binds to or significantly degrades the enzyme so that its function cannot recover. Nerve gas and penicillin both make use of this type of inhibition, albeit with massively different intentions in mind.

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