6 Key excerpts on "Enzyme Activity"

• 

  eBook - ePub

  Introduction to Nutrition and Metabolism

  • David A Bender, Shauna M C Cunningham(Authors)
  • 2021(Publication Date)
  • CRC Press

    (Publisher)

  Section 2.3.3.3 ) that show cooperative binding of substrate. Binding substrate at one of the binding sites affects the conformation at the other active sites, enhancing the binding of further molecules of substrate.

  2.2.3 Units of Enzyme Activity

  In relatively rare cases when an enzyme has been purified, it is possible to express the amount of the enzyme in tissues or plasma as the number of moles of enzyme protein present, for example, by raising antibodies against the purified protein for use in an immunoassay. However, what is more important is not how much of the enzyme protein is present in the cell, but rather how much catalytic activity there is – how much substrate can be converted to product in a given time. Therefore, the amount of enzymes is usually expressed in units of activity.

  The SI unit of catalysis is katal = 1 mol of substrate converted per second. However, Enzyme Activity is usually expressed as the number of micromoles (µmol) of substrate converted (or of product formed) per minute. This is the standard unit of Enzyme Activity, determined under specified optimum conditions for that enzyme, at 30°C. This temperature is a compromise between mammalian biochemists, who would work at body temperature (37°C for human beings), and microbiological biochemists, who would normally work at 20°C.

  2.3 Factors Affecting Enzyme Activity

  Any given enzyme has an innate activity – for many enzymes the catalytic rate constant is of the order of 1000–5000 mol of substrate converted per mol of enzyme per second or higher. However, a number of factors affect the activity of enzymes.

  2.3.1 The Effect of pH

  Both the binding of the substrate to the enzyme and catalysis of the reaction depend on interactions between the substrates and reactive groups in the amino acid side chains that make up the active site. They have to be in the appropriate ionization state for binding and reaction to occur, and this depends on the pH of the medium. Any enzyme will have maximum activity at a specific pH – the optimum pH for that enzyme. If the pH rises above or falls below the optimum, then the activity of the enzyme will decrease. Most enzymes have little or no activity at a pH of 2–3 units away from their optimum. Although the average pH of cell contents (and plasma) is around 7.4, individual subcellular compartments and organelles may be very acidic or alkaline.

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  eBook - ePub

  Medical Biochemistry

  • Antonio Blanco, Gustavo Blanco(Authors)
  • 2017(Publication Date)
  • Academic Press

    (Publisher)

  transition intermediary , which will be subjected to catalysis.

  Zymogens or proenzymes are inactive precursors of enzymes. They acquire activity after hydrolysis of a portion of their molecule.

  Cellular location of enzymes varies, the majority being in different compartments of the cell, while others are extracellular.

  Multienzyme systems are those composed of a series of enzymes or enzyme complexes. There are also multifunctional enzymes with several different catalytic sites in the same molecule.

  Enzyme Activity is determined by measuring the amount of product formed, or substrate consumed in a reaction in a given time. Initial velocity corresponds to the activity measured when the amount of consumed substrate is less than 20% of the total substrate originally present. One IU of enzyme catalyzes the conversion of 1 μmol of substrate per second under defined conditions of pH and temperature. Specific activity is the units of enzyme per milligram of protein present in the sample. Molar activity or turnover number are the substrate molecules converted into product per unit time per enzyme molecule, under conditions of substrate saturation.

  The rate of the enzymatic reaction is directly proportional to the amount of enzyme rate present in the sample.

  Also, at low [S] and under constant conditions of the medium, Enzyme Activity rapidly increases with the raise in [S]. At higher substrate levels, the activity increases slowly and tends to reach a maximum. The effect follows a hyperbolic

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  eBook - ePub

  Pharmaceutical Biotechnology

  A Focus on Industrial Application

  • Adalberto Pessoa, Michele Vitolo, Paul Frederick Long, Adalberto Pessoa, Michele Vitolo, Paul Frederick Long(Authors)
  • 2021(Publication Date)
  • CRC Press

    (Publisher)

  Enzymes are catalytic proteins capable to convert a compound (substrate) to another (product) at high reaction rate. In enzyme-catalyzed reactions, enzymes, in general, specifically accept only one substrate. Even when an enzyme catalyzes the conversion of more than one substrate, the reaction rates are different. Substrate specificity is linked to a region of the enzyme molecule called the active site, which can be envisaged as a rigid or flexible structure depending on the particular reaction considered. Currently, it is considered that the active site is organized into two sub-regions. At one of them, the substrate fitting occurs (the so-called bonding site), and in the other, the chemical transformation of the substrate occurs (the so-called catalytic site). Generally, it can be considered that an enzyme-catalyzed reaction proceeds through three different steps: first step (period in which the enzyme and substrate are mixed into the reaction medium. The enzyme-substrate complex is formed and accumulates); second step (a period during which the concentration of the ES complex remains constant in the reaction medium, the substrate concentration diminishes, and the product concentration increases); third step (the concentration of the ES complex in the reaction medium is not constant; the consumption of substrate and formation of product occur more slowly). The fundamental aspect is that the catalysis rate and substrate concentration follow a hyperbolic correlation. The Enzyme Activity can be affected by factors of different nature, i.e., physical-chemical factors (pH, temperature, ionic strength, water activity, etc.), chemical factors (activators, inhibitors –irreversible or reversible (competitive, non-competitive and uncompetitive) –, stabilizers, and deactivators) and physical factors (pressure and shear forces). The ability to quantify Enzyme Activity is essential if an enzyme is to be used in a cost-effective way in an industrial process or other analytical methods. Focusing on industrial processes, some considerations must be made in order to evaluate the suitability using enzymes as catalysts in specific reactions. The basic considerations should be: (a) the amount of enzyme necessary; (b) the duration of the reaction; (c) the initial substrate concentration necessary; (d) the reaction conditions; and (e) the cost involved. In general, the effective cost of an enzymatic process derivates from the ratio between the additional cost due to the enzyme and the improvement of the process yield and/or the aggregate value of the final product, taking into consideration the economic cost of the overall process.

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  eBook - ePub

  Chemistry of Biomolecules, Second Edition

  • S. P. Bhutani(Author)
  • 2019(Publication Date)
  • CRC Press

    (Publisher)

    3  

  Enzymes

  Learning Objectives In this chapter we will study •  Enzymes – Nomenclature and Classification •  Characteristics of Enzymes – Catalytic Power, Enzyme Specificity and Stereo-specificity •  Factors Influencing Enzyme Activity – Effect of Temperature and pH on Enzyme Activity; Effect of Substrate and Enzyme Concentration •  Cofactors and Coenzymes

  •  Some Important Coenzymes – NAD+ , NADP+ , Coenzyme A, Pyridoxal-5-phosphate, FMN, FAD, TPP

  •  Enzyme Inhibitors •  Mechanism to Enzyme Catalysis – Lock and Key Model •  Alcoholic Fermentation •  Glycolysis •  Citric Acid Cycle •  Introduction to Green Chemistry •  Biocatalysis–Importance in Green Chemistry and Chemical Industry

  3.1    INTRODUCTION

  We consume various food ingredients - carbohydrates, proteins and fats - any time we take our meals. These are digested and give us energy for performing the various metabolic functions in our body. The secret ingredient in living organisms is catalysis. The catalysts in living cells which facilitate the metabolic reactions are called enzymes. The miracle of life is that chemical reactions in the cell occur with great accuracy and at astonishing speed. Without the proper enzymes to process the food we eat, it might take us years to digest our breakfast or lunch.

  With the exceptions of some RNAs that have catalytic activity, all enzymes are proteins or their derivatives and vary in molecular weight from 10,000 up to 5,00,000. Of all the functions of proteins, catalysis is probably the most important. In the absence of catalysis, most reactions in biological systems would take place too slowly to provide products at an adequate speed for a metabolising organism. A number of enzymes have been isolated and obtained in a crystalline form. Other enzymes are derivatives of proteins formed by combination with some other group such as a metal. Enzymes containing a wide range of metals including iron, copper, zinc, manganese and magnesium are known. In other cases, the protein is combined with a non-proteinous organic molecule. We shall study these under cofactors.

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  eBook - ePub

  How Enzymes Work

  From Structure to Function

  • Haruo Suzuki(Author)
  • 2019(Publication Date)
  • Jenny Stanford Publishing

    (Publisher)

  Chapter 3

  Factors That Affect Enzyme Activity

  The previous chapters described briefly that the Enzyme Activity is affected by the concentration of substrate and of product. This and the following chapters describe the effect of factors affecting the Enzyme Activity more in detail. What can we get by studying the effect of various factors on the activity of enzyme? These studies will lead to a construction of the reaction mechanism, and will give us the thermodynamic parameters of the reaction.

  3.1    Enzyme Concentration

  In Chapter 2 , we have known that the enzymatic reaction is expressed by the Michaelis–Menten mechanism:

  E + S ⇄ k − 1 k + 1     ES → k + 2 E + P

  (3.1)

  Under the steady state of reaction, the rate of the reaction is expressed by the Michaelis–Menten equation:

  v = k + 2 e 0 s K m + s

  (3.2)

  K m

  =

  k

  − 1

  +

  k

  + 2

  k

  + 1

  Thus, as Eq. 3.2 shows, plotting v vs. the enzyme concentration gives a linear relation (line A in Fig. 3.1 ) when the concentration of substrate is sufficiently larger than that of the concentration of enzyme.

  Figure 3.1 The effect of the concentration of enzyme on the enzymatic activity.

  Sometimes, the data like lines B and C will be obtained. In these cases, the Michaelis–Menten kinetics could not be applied. At least two cases are conceivable when these data were obtained. One is artifacts caused by the experimental conditions, and the other is the nature of the enzyme itself. In the first case, the enzyme preparation and/or the reaction mixture may contain an inhibitor or an activator of enzyme. In the case of line B, the enzyme preparation may contain an activator of enzyme, but the activator may dissociate at the low concentration of the enzyme. Or the reaction mixture may contain an inhibitor of enzyme, thus inhibiting at low concentration of enzyme. However, high concentrations of enzyme consume the inhibitor and prevent the inhibition. Similarly, in the case of line C, the enzyme preparation may contain the inhibitor of enzyme, thus the inhibitor dissociates from the enzyme at the low concentration of enzyme, showing relatively high activity. Or the reaction mixture may contain the activator of the enzyme, leading line C. In these cases, removal of the inhibitor or activator in the reaction mixture including enzyme itself may cause the linearity in the plot of the activity vs. enzyme concentration. Even though an inhibitor or an activator was removed, the enzyme still gives the data like lines B or C. This means that the enzyme has its own nature to show such a result. These considerations lead the important conclusion that the kinetic experiment must be performed using the highly purified enzyme.

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  eBook - ePub

  Fundamentals of Enzyme Kinetics

  • Athel Cornish-Bowden(Author)
  • 2013(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  Chapter 11 Temperature Effects on Enzyme Activity

  11.1 Temperature denaturation

  In principle, the theoretical treatment discussed in Section 1.8 of the temperature dependence of simple chemical reactions applies equally to enzyme-catalyzed reactions, but in practice there are several complications that must be properly understood if any useful information is to be obtained from temperature-dependence measurements.

  § 1.8, pages 15–21

  First, almost all enzymes become denatured if they are heated much above physiological temperatures, and the conformation of the enzyme is altered, often irreversibly, with loss of catalytic activity. Denaturation is chemically a complicated and only partly understood process, and only a simplified account will be given here. In this section I shall limit it to reversible denaturation, assuming that an equilibrium exists at all times between the active and denatured enzyme and that only a single denatured species needs to be taken into account. However, I emphasize that limiting it to reversible denaturation is for the sake of simplicity, not because irreversible effects are unimportant in practice.

  Denaturation does not involve rupture of covalent bonds, but only of hydrogen bonds and other weak interactions that are involved in maintaining the active conformation of the enzyme. Although an individual hydrogen bond is far weaker than a covalent bond (about 20 kJ · mol−1 for a hydrogen bond compared with about 400 kJ · mol−1 for a covalent bond), denaturation generally involves the rupture of many of them. More exactly, it involves the replacement of many intramolecular hydrogen bonds with hydrogen bonds between the enzyme molecule and solvent molecules. The standard enthalpy of reaction, ΔH 0 ′, is often very high for denaturation, typically 200–500 kJ · mol−1 , but the rupture of many weak bonds greatly increases the number of conformational states available to an enzyme molecule, and so denaturation is also characterized by a large standard entropy of reaction, ΔS 0

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