Biological Sciences
Factors Affecting Enzyme Activity
Factors affecting enzyme activity include temperature, pH, substrate concentration, and the presence of inhibitors or activators. Enzymes function optimally within specific temperature and pH ranges, and their activity is influenced by the concentration of substrate molecules. Inhibitors can decrease enzyme activity, while activators can enhance it. These factors collectively impact the efficiency of enzyme-catalyzed reactions.
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eBook - PDFChemistry for Today
General, Organic, and Biochemistry
- Spencer Seager, Michael Slabaugh, Maren Hansen, , Spencer Seager, Spencer Seager, Michael Slabaugh, Maren Hansen(Authors)
- 2021(Publication Date)
- Cengage Learning EMEA(Publisher)
For example, an enzyme preparation with an activity corresponding to 40 IU contains a concentration of enzyme 40 times greater than the standard. This is a useful way to mea- sure enzyme activity because the level of enzyme activity compared with normal activity is significant in the diagnosis of many diseases (see Section 20.9). Example 20.2 Describing Enzyme Activity List two ways of describing enzyme activity. Solution Turnover number and enzyme international unit. ✔ LEARNING CHECK 20.2 Differentiate between the terms turnover number and enzyme international unit. 20.6 Factors Affecting Enzyme Activity Learning Objective 6 Identify the factors that affect enzyme activity. Several factors affect the rate of enzyme-catalyzed reactions (see Figure 20.7). The most important factors are enzyme concentration, substrate concentration, temperature, and pH. In this section, we’ll look at each of these factors in some detail. In Section 20.7, we’ll consider another very important factor, the presence of enzyme inhibitors. 20.6A Enzyme Concentration In an enzyme-catalyzed reaction, the concentration of enzyme is normally very low com- pared with the concentration of substrate. When the enzyme concentration is increased, the concentration of ES also increases in compliance with reaction rate theory: E 1 S S d ES increased [E] gives more [ES] Thus, the availability of more enzyme molecules to catalyze a reaction leads to the for- mation of more ES and a higher reaction rate.
eBook - PDF- Osei, Getachew(Authors)
- 2018(Publication Date)
- Agri Horti Press(Publisher)
Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5°C or below is generally the most suitable. Some enzymes lose their activity when frozen. Effects of pH Enzymes are affected by changes in pH. The most favourable pH value-the point where the enzyme is most active-is known as the optimum pH. Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. The optimum pH value will vary greatly from one enzyme to another, table shows: This ebook is exclusively for this university only. Cannot be resold/distributed. 110 Industrial Microbiology and Biotechnology Table. pH for Optimum Activity. Enzyme pH Optimum Lipase (pancreas) 8.0 Lipase (stomach) 4.0-5.0 Lipase (castor oil) 4.7 Pepsin 1.5-1.6 Trypsin 7.8-8.7 Urease 7.0 Invertase 4.5 Maltase 6.1-6.8 Amylase (pancreas) 6.7-7.0 Amylase (malt) 4.6-5.2 Catalase 7.0 In addition to temperature and pH there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimised in order for an enzymatic reaction to be accurate and reproducible. The term enzyme kinetics implies a study of the speed, rate or velocity of an enzyme catalysed reaction, and of the various factors which may affect this. At the heart of any study of enzyme kinetics is a knowledge of the way in which reaction velocity is altered by changes in the concentration of the enzyme’s substrate and of the simple mathematics underlying this. To ease ourselves gently into this as suggested, assume that the enzyme that we are discussing has no special features, such as allosteric properties, and catalyses the conversion of just one substrate to one product.
eBook - PDFMicrobial Engineering
First International Symposium on Advances in Microbial Engineering
- Z. Sterbacek(Author)
- 2016(Publication Date)
- Butterworth-Heinemann(Publisher)
THE INFLUENCE OF ENVIRONMENTAL FACTORS ON THE KINETICS OF A BIOSYNTHETIC PROCESS L. A. MUZYCHENKO 1 , V. M. KANTERE 2 and V. A. GURKIN 1 1 All-Union Research Institute of Genetics and Selection of Industrial Microorganisms, 2 Institute of Chemical Equipment Construction, Moscow, USSR ABSTRACT The efforts of specialists in the kinetics of biosynthetic processes are con-centrated on the investigation of kinetic relations depending on a limiting stage of the process (diffusive or kinetic). If a kinetic study is needed, then the kinetics of the process in relation to the limiting component (substrate or a metabolic product) is investigated. The influence of environmental factors, whether thermodynamic (temperature, pressure) or parametric (pH and oxidation-reduction potential), is usually not considered. Investigations were carried out on the influence of temperature on the rate of formation and decay of an enzyme using the Arrhenius equation, on the influence of pH enzyme dissociation and on substrate rate of formation and decay in different stages of substance transformation into the cell material. These relations were generalized by a mathematical dependence of the growth rate on environmental temperature and pH value. INTRODUCTION Increased capacities of microbiological industries have resulted in a strong interest in the optimization of microorganism culture conditions. The whole set of parameters influencing the processes of microbiological syn-thesis can be divided into two groups. The first group covers the substance concentrations used by micro-organisms while the second one comprises the so-called physical as well as physicochemical factors: pH, eH, temperature, and pressure. Poorly-soluble substances injected by gas stream, e.g. oxygen, can also be included here, since their concentration in the medium not only depends on their partial pressure in the gas stream but also on physical and physicochemical factors which determine the solubility of gas components.
eBook - PDF- BIOTOL, B C Currell, R C E Dam-Mieras(Authors)
- 2013(Publication Date)
- Butterworth-Heinemann(Publisher)
Enzymes 8.1 Introduction 186 8.2 Introduction to enzymatic catalysis 186 8.3 The assay of enzymes 201 8.4 Effect of substrate concentration on enzyme activity 210 8.5 Effect of pH and temperature on enzyme activity 218 8.6 Effect of inhibitors on enzyme activity 222 8.7 Effect of cofactors 232 8.8 Mechanisms of enzymatic catalysis 235 8.9 Allosteric enzymes and regulation of enzyme activity 238 8.10 Other methods of regulating enzyme activity 246 8.11 Enzyme classification 248 8.12 Enzyme technology 250 Summary and objectives 254 185 186 Chapter 8 Enzymes 8.1 Introduction In Chapter 1 we described cells as behaving like superb chemical factories, converting one set of chemicals (nutrients or substrates) into a vast number of complex products. We also described cells as being able to bring about these changes quickly and efficiently at a cool temperature. In subsequent chapters we examined many of the products of these chemical factories. We have learnt that many of the these are chemically very complex and are of specified stereochemical configuration. For example we have learnt that amino acids are produced in the L-configuration. The ability of cells to carry out these chemical processes in such an efficient and specific manner undoubtedly depends upon the structural organisation of cells and on their ability to produce suitable catalysts for speeding up the desired reactions. We met with one aspect of the structural organisation of cells at a molecular level in Chapter 7, when we discussed the structure and properties of membranes. In this chapter, we examine the catalysts used by cells. These catalysts are all proteins and are called enzymes. This chapter is a long one, reflecting the importance that must be attached to enzymes if we are to understand how living systems can bring about the vast array of chemical changes in a controlled and predictable manner. Do not attempt to study all of this chapter in one sitting.
eBook - ePub- S. P. Bhutani(Author)
- 2019(Publication Date)
- CRC Press(Publisher)
e.g., enzymes of various species of bacteria inhabiting hot springs are active at temperatures exceeding 85°C. Some enzymes, such as ribonuclease lose activity on heating but quickly regain it on cooling indicating that their unfolded polypeptide chain quickly reverts back into its natural conformation.B. Effect of pH on Enzyme Activity
The symbol pH refers to the concentration of hydrogen ions in solution. The activity of an enzyme varies with the pH of the medium. Most enzymes have a characteristic pH at which their activity is maximum. Above or below this pH the activity declines. When we plot activity versus pH most enzymes yield a bell-shaped curve with a more or less sharply defined maximum. That means enzymes have maximum activity at optimum pH. Figure 3.2 illustrates the relationship between the pH and activity of the enzyme invertase.We give below in Table 3.2 , the optimum pH for some typical enzymes. The optimum pH of an enzyme is not ncessarily identical with the pH of its normal intracellular surroundings.Even small changes in pH can have a great effect on enzyme activity. Small changes in pH mean relatively large changes in [H+ ]. A change of 1 on the pH scale involves a ten-fold increase or decrease in [H+ ], while a change in pH of 2 represents a hundred-fold change in [H+ ]. The concentration of [H+ ] affects the stability of the electrovalent bonds which help to maintain the tertiary structure of protein molecules. Extremes of pH cause the bonds to break resulting in enzyme denaturation.Fig. 3.2 Relationship between the pH and activity of the enzyme.The affinity of an enzyme for its substrate may be altered by variations in pH. Changes in [H+ ] can alter the ionisation of the amino acid side chains at the active centres of enzymes. Ionisation of the substrate molecules can also be affected. The formation of enzyme-substrate complexes depends on the active centres and substrate molecules having opposite electrostatic charges. If the charges are altered by changes in pH, some enzymes fail to function.TABLE 3.2. Optimum pH for Some Enzymes
eBook - ePub- Athel Cornish-Bowden(Author)
- 2013(Publication Date)
- Wiley-Blackwell(Publisher)
Chapter 10 Effect of pH on Enzyme Activity10.1 Enzymes and pH
Of the many problems that beset the first investigators of enzyme kinetics, none was more important than the lack of understanding of hydrogen-ion concentration, [H+ ]. In aqueous chemistry, [H+ ] varies from about 1 M to about 10−14 M, an enormous range that is commonly decreased to more manageable proportions by the use of a logarithmic scale, pH = − log [H+ ]. All enzymes are profoundly influenced by pH, and no substantial progress could be made in the understanding of enzymes until Michaelis and his collaborators made pH control a routine part of all serious enzyme studies. The concept of buffers for controlling the hydrogen-ion concentration, and the pH scale for expressing it, were first published by Sørensen, in a classic paper1 on the importance of hydrogen-ion concentration in enzyme studies. Michaelis, however, was already working on similar lines, and it was not long afterwards that the first of his many papers on effects of pH on enzymes appeared, written with Davidsohn. Although there are still some disagreements about the proper interpretation of pH effects in enzyme kinetics, the practical importance of pH continues undiminished: it is hopeless to attempt any kinetic studies without adequate control of pH.SØREN PETER LAURITZ SØRENSEN (1868–1939) was born near Slagelse (Denmark), and studied first medicine and then chemistry in Copenhagen. He made his career at the Carlsberg Laboratories, where he was particularly interested in amino acids and proteins. The effect of temperature on enzyme activity was already reasonably well understood, but Sørensen realized that this was far from being the only factor, and that there was an urgent need for developing methods for measuring and controlling the concentration of hydrogen ions.It may seem surprising that it was left to enzymologists to draw attention to the importance of hydrogen-ion concentration and to introduce the use of buffers. We may reflect, therefore, on the special properties of enzymes that made pH control imperative before any need for it had been felt in the already highly developed science of chemical kinetics. With a few exceptions, such as pepsin and alkaline phosphatase, the enzymes that have been most studied are active only in aqueous solution at pH values in the range 5–9. Indeed, only pepsin has a physiologically important activity outside this middle range of pH. Now, in the pH range 5–9, the hydrogen-ion and hydroxide-ion concentrations are both in the range 10−5 –10−9
eBook - PDF- Sudheer Awasthi(Author)
- 2023(Publication Date)
- Arcler Press(Publisher)
Figure 5.11. Demonstrates that the speed of the majority of enzymatic pro- cesses about doubles for each 10oC increase in temperature. Source: https://commons.wikimedia.org/wiki/File:Effect_of_temperature_on_ enzymes.svg Introduction to Enzymology 176 370 degrees Celsius is the ideal condition for an enzyme in the human body, and the Q10 for enzyme-catalyzed reactions is 2. Even though the majority of enzymes are inactive at temperatures over 55oC, some are highly stable and sustain activity at considerably higher temperatures; for instance, the enzymes of several kinds of bacteria residing in hot springs are active at 85oC. Many enzymes, like ribosomes, lose their action when heated, but recover it when cooled, showing that the unfolded polypeptide chain rapidly returns to its native conformation. 5.7.2. Effect of pH on enzyme activity The logo pH corresponds to the hydrogen ion density in a solution. The activity of the enzyme alters the pH of the solution. Most enzymes get a pH during which their action is at its peak. Up or down this pH, the action begins to decline (Tien & Kirk, 1984). Once we arrange action vs. pH, the majority of enzymes exhibit a Bell-shaped contour with a rather strongly defined peak, indicating that the activity of the enzyme is highest at the optimal pH. Figure 5.12. Narrow pH variations could have a significant impact on enzy- matic activity. Source: https://commons.wikimedia.org/wiki/File:Effect_of_pH_on_enzymes. svg Characterization of Enzymes 177 Variations in pH have a significant impact on hydrogen ion density. A fluctuation of 1 on the pH scale corresponds to a tenfold up or down in the potential of hydrogen, whereas a difference of 2 corresponds to a hundredfold variation in the number of hydrogen ions (Garen & Levinthal, 1960). The density of hydrogen ions improves the reliability of the electrovalent linkages that support the protein molecule’s tertiary structure.
eBook - PDF- David Ucko(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
But just as increasing the substrate concentration favors the formation of more product, the buildup of product causes an increase in the reverse reac-tion and a s l o w d o w n in the forward rate o f reaction. T h e temperature generally increases the rate of reaction b y about 1.1 to 3 times for every rise of 10°C. But enzymes have an optimum temperature, at which their activity is greatest. T h e normal b o d y temperature, 37°C (98.6°F), is near this b e s t temperature for most enzymes. Since they are proteins, en-zymes denature at high temperatures, such as at 60°C or above. E n z y m e activity also depends on the pH of the surrounding fluid. T h e activ-ity is greatest at an optimum p H , which is often b e t w e e n p H 6 and 8. A no-table exception is pepsin, the enzyme of the gastric juice in the stomach. Be-cause the optimum p H of pepsin is about 2, it functions effectively in this acidic fluid. n e a t rs Some enzymes n e e d an extra nonprotein part, called a cofactor, for their activity. Neither the protein part alone (the apoprotein) nor the cofactor alone can act as catalyst in these cases; only their combination (the holoenzyme) can serve this function. W h e n the cofactor is firmly b o u n d to the protein, it is known as a prosthetic group. If the cofactor is an organic molecule, it is called a c o e n z y m e ; as described in Chapter 21 many o f the vitamins you eat b e c o m e coenzymes. Some ex-amples of coenzymes and their functions are listed in Table 16-2. Coenzymes often transfer atoms or electrons to or from the substrate molecule. T h e y are discussed in greater detail in the next chapter.
eBook - ePubContemporary Enzyme Kinetics and Mechanism
Reliable Lab Solutions
- (Author)
- 2009(Publication Date)
- Academic Press(Publisher)
Chapter 6 Effects of pH on Enzymes*Keith F. Tipton, Andrew G. McDonald Henry B.F. Dixon✠ , Department of Biochemistry, Trinity College, Dublin 2, Ireland- Abstract
- I. Theory
- A. The Ionization of Dibasic Acids (Adams, 1916 )
- B. Simplified System
- C. Methods of Obtaining Ionization Constants
- D. Complications
- E. Interpretation of the Results of pH Experiments
- F. pH-Independence of K m
- G. Identification of Amino Acid Residues from Their Ionizations
- II. Limitations of the Methods
- III. Practical Aspects
- A. Effects of pH on the Stability of Enzymes
- B. Effects of pH on Substrates
- C. Effects of pH on the Assay Method
- D. Buffers
- IV. Some Examples of pH Studies
- A. Carnitine Acetyltransferase
- B. Fumarate Hydratase
- C. Lysozyme
- V. Future Prospects
- References
The effects of variations of the hydrogen ion concentration on the activity of enzymes have close similarities to the effects of activators and inhibitors, and the same kinetic methods and theory can be applied to both types of system. These close similarities are often obscured, however, by the fact that a logarithmic scale (pH) is usually used for hydrogen ion concentration. Treatment of the effects of hydrogen ion concentration in the same way as other effectors can yield valuable information on the nature of the kinetic mechanism obeyed by the enzyme; in addition, the characteristic ionization constants of amino acid side-chain groups has led to the use of such studies in attempts to identify specific groups as playing a role in the reaction. This latter approach has frequently been regarded as being the most important function of these studies. These two aspects of the subject, however, are complementary, and attempts to identify groups from pH studies without considering the kinetic aspects not only will miss a great deal of valuable information, but also can frequently lead to erroneous conclusions. In this chapter, we will consider the effects of hydrogen ion concentration on the activity of enzymes both in terms of kinetic analysis with the hydrogen ion concentration as the variable and in terms of the use of the effects of pH to identify specific ionizing groups.
eBook - PDF- P. Tijssen(Author)
- 1985(Publication Date)
- Elsevier Science(Publisher)
The rate of an enzyme-catalyzed reaction as a function of pH generally yields a bell-shaped curve. Some enzymes are very sensitive to small changes in pH (lysozyme), whereas others (POase) are rela- tively insensitive (within 1-2 pH units near their optima). Enzymes with similar activities but from different origin may have very differ- ent optima. APase from Escherickia coli is optimally active at a pH of about 8, whereas APase from calf intestine is most active around pH 10 and the activity of these enzymes decreases strongly outside their optima. Nevertheless, the E. coli enzyme is often assayed at the pH optimum of the intestinal enzyme. The optimum substrate concentrations may also be pH dependent for some enzymes (Chapter 10). Temperature effects can be either negative or positive on the K , or the kcat of the reaction but can only be negative on the enzyme (denaturation). If K,,, = Ks, K , may be determined at various temper- atures. The k,,, can be established directly from the Arrhenius equa- tion (eq. l): U In k,,, = In Z - - RT plotting In kcltvs. 1/T yields a straight line with a slope of -a/R. 164 PRACTICE & THEORY OF ENZYME IMMUNOASSAYS An increase of 1°C in the temperature may enhance the reaction rate by more than 10% until the optimum. Thereafter the enzyme is inactivated. The standard temperature for the measurement of enzyme activity is 30°C, though 25°C or 37°C have also been used. The pH optimum for an enzyme may shift with the temperature: APase has an pH optimum of 10.3, 10.1 and 9.9 at 25, 30, and 37C, respectively. The preferred temperature for POase is around 15°C; though it may initially be more active at a somewhat higher temperature, it is relatively faster inactivated. The addition of a non-ionic detergent delays this inactivation and higher temperatures may be used (Section 10.1.1.4.2). Buffers may influence the kinetic properties of enzymes in a number of ways.
eBook - PDF- M Volkenstein(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
Molecular activity can also be expressed as the turnover rate (Warburg), which is the number of moles of substrate transformed per mole of enzyme per minute. Methods for extracting and purifying enzymes are well ad-vanced, and many enzymes have been obtained in the pure crystal-line form [15,30,31]. Thus it is possible to study reactions and structures of enzymes in vitro. The situation in vitro dif-fers from that in the cell because the cell is an open system and à great number of enzymic reactions, including coupled re-actions, proceed there simultaneously. However, the study of enzymes in vitro provides a strong foundation for understanding the corresponding biological processes. When an ESC (enzyme-substrate complex) is formed, the small substrate molecule (or molecules) is (are) bound stoichiometri-cally to a large enzyme molecule. Evidently the substrate binds directly to some specific small region of the enzyme molecule, called the active site. The nature of the active site, that is, the nature of the amino acid residues and their positions, as well as of the cofactors, participating at the active site, can be established by chemical and physical methods. The altera-tions in activity produced by chemical modifications of a pro-tein make it possible to determine the functional groups of the active site. Information about its structure can be obtained by means of spectrophotometry, spectropolarimetry, NMR and EPR spectroscopy (in the latter a paramagnetic label is introduced), etc. X-ray structural analysis reveals the actual geometric pattern of enzyme-substrate interaction. The diversity of amino acid residues and atomic groups of cofactors determines the polyfunctional properties of the active site, its ability to bind molecules of the substrate or modifier, and the catalytic activity [31].
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