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If there is a magical component to life, an argument can surely be made for it being catalysis. Thanks to catalysis, reactions that could take hundreds of years to complete in the “real world," occur in seconds in the presence of a catalyst. The secret to catalytic action is reducing the magnitude of that barrier, as we shall see.
Introduction to Enzyme and Coenzyme Chemistry, 3rd Edition
Enzymes are giant macromolecules which catalyse biochemical reactions. They are remarkable in many ways. Their three-dimensional structures are highly complex, yet they are formed by spontaneous folding of a linear polypeptide chain. Their catalytic properties are far more impressive than synthetic catalysts which operate under more extreme conditions. Each enzyme catalyses a single chemical reaction on a particular chemical substrate with very high enantioselectivity and enantiospecificity at rates which approach &ldquocatalytic perfection&rdquo. Living cells are capable of carrying out a huge repertoire of enzyme-catalysed chemical reactions, some of which have little or no precedent in organic chemistry.
The popular textbook Introduction to Enzyme and Coenzyme Chemistry has been thoroughly updated to include information on the most recent advances in our understanding of enzyme action, with additional recent examples from the literature used to illustrate key points. A major new feature is the inclusion of two-colour figures, and the addition of over 40 new figures of the active sites of enzymes discussed in the text, in order to illustrate the interplay between enzyme structure and function.
This new edition provides a concise but comprehensive account from the perspective of organic chemistry, what enzymes are, how they work, and how they catalyse many of the major classes of enzymatic reactions, and will continue to prove invaluable to both undergraduate and postgraduate students of organic, bio-organic and medicinal chemistry, chemical biology, biochemistry and biotechnology.
Properties of Life
- Evolution: long term adaptation/speciation
- Cell-based: Cells are the fundamental unit of life
- Complexity: allows physical/biochemical changes (dynamic order)
- Homeostasis: maintains balance between change and order
- Requires Energy: needed to do work (cellular functions)
- Irritability: immediate sensitivity and response to stimuli
- Reproduction: the ability to propogate life
- Development: programmed change, most obvious in multicellular organisms
Remember, to be alive is to possess not just some, but all of these properties! If entities with all of the properties of life (i.e., cells) did originate independently, they would have reproduced to form separate populations of cells. In this scenario, less successful populations go extinct while successful ones become dominant. Successful organisms would have spread, spawning populations and generating new species. The take-home message is that if conditions on a prebiotic earth favored the formation of the ‘first cell’, then why not the formation of two, or dozens or even hundreds of ‘first cells’? However, we will see that only one successful population of cells would survive to become the source of the common ancestor of all life on earth, while other populations became extinct.
As to the question of when life began, geological and geochemical evidence suggests the presence of life on earth as early as 4.1 billion years ago. As for how life began, this remains the subject of ongoing speculation. All of the scenarios described below attempt to understand the physical, chemical and energetic conditions that might have been the ideal laboratory for prebiotic “chemistry experiments”. What all the scenarios share are the following requirements.
Structure and Function of Cytochrome P450 Enzymes
Frederick P. Guengerich , in Reference Module in Life Sciences , 2020
Reaction Chemistry in Cytochrome P450 Enzymes
Catalysis by cytochrome P450 enzymes is understood largely in the context of an intermediate called Compound I, a term developed in work with the related peroxidase enzymes. Each cycle of catalysis involves a series of electronic changes in the central iron atom (RH: substrate, ROH: product) ( Fig. 2 ) ( Ortiz de Montellano, 2015 Guengerich, 2018 ).
Fig. 2 . General catalytic cycle for cytochrome P450 reactions. The steps include: (1) substrate (RH) binding (near the iron atom of the heme prosthetic group, but not bonding to the iron itself (2) 1-electron reduction of the iron atom by an accessory protein (diflavin reductase or a ferredoxin) (3) addition of molecular oxygen (O2) (4) introduction of a second electron into the Fe 2+ O2 complex. (from a diflavin reductase or a ferredoxin, or the hemoprotein cytochrome b5) protonation of the (formal) Fe 3+ –O2 – complex (5) scission of the “Compound 0” complex to form FeO 3+ (Compound I) (6) abstraction of a hydrogen atom (or non-bonded electron) from the substrate (RH) (7) “oxygen rebound” to the incipient radical formed from the substrate, generating the product (ROH) (8) release of the product (ROH). Reproduced from Ortiz de Montellano, P.R., 2015. Substrate oxidation. In: Ortiz de Montellano P.R. (Ed.), Cytochrome P450: Structure, Mechanism, and Biochemistry, fourth ed. New York: Springer, pp. 111–176. Guengerich, F.P., 2018. Perspective: Mechanisms of cytochrome P450-catalyzed oxidations. ACS Catalysis 8, 10964–10976.
All of the intermediates following the addition of the first electron are unstable, especially after the addition of O2. Compound I has required specialized methods for its detection and has been observed only in the case of a few (four) bacterial cytochrome P450 enzymes and probably mammalian cytochrome P450 11A1. It is a very highly oxidized iron-oxygen species and reacts with alkanes with a lifetime of only 30 ms. Further, the next step (recombination) is also rapid, yielding the product.
The role of the protein structure is several fold in cytochrome P450 enzymes. One role is to bind each substrate in such a way as to facilitate catalysis at a particular site. The other is to facilitate the steps leading to formation of Compound I. These two functions are often intertwined. For instance, the binding of a substrate to a cytochrome P450 can facilitate the ease of reduction by enhancing the binding of an auxiliary electron transfer protein or by lowering the oxidation potential of the iron atom in some cases ( Fig. 2 ). The substrate can also stabilize some of the intermediates, e.g., the Fe 2+ O2 complex.
Although the substrate radicals produced by the Compound I form of a cytochrome P450 are unstable, in some cases these can rearrange before the “rebound” of the oxygen, yielding unexpected products ( Fig. 1 ). As mentioned earlier, two substrate radicals can even combine to form new C–C or other bonds ( Fig. 1(B) and (C) ). These reactions are guided by elements of the protein structure, although the details underlying specific migrations are not well understood at a molecular level.
4.1: Introduction to Catalysis - Biology
I590: Introduction to genomics for non-biologists (3CR)
Spring Semester 2005: Tuesday/Thursday, 9:30am-10:45pm , Eigenmann 921
Description: We aim to introduce the broad frontiers of contemporary biology to the students who intend to work on biology-related problems but so far have no biology background. This class will cover important themes in molecular genetics and cell biology, including
- biochemistry of proteins and nucleic acids
- 3D structure of biological molecules
- genetic mechanisms
- internal organization of the cell: membrane, intracellular compartments and traffic, and the cell cycle
- cell communiation
- immune system
- development of multicellular organisms
- biotechnologies in genomics and proteomics.
This course is designed for non-biology-major graduate students working in the biology-related areas, particularly bioinformatics and computational biology. Therefore, we assume the students are mature students who want to learn biology contents as quickly as possible so they can return to their biology-related projects and collabrate with biologists smoothly. We expect after taking this class, the students will be able to (1) understand the main idea in biological literatures (2) communicate with biologists with no difficulty (3) write the summary of the biological essence in their biology-related projects without much help from biology collabrators.
Textbook: Alberts, Johnson, Lewis, Raff, Roberts, Walter: Molecular biology of the cell, 4th edition. This is an expensive book and we mainly use it as a reference. However, if you intend to continue working on biology-related problems, I recommend you buy this excellent textbook. Certainly you can also refer this book from its online version at NCBI website with a text searching fashion: Some of the topics from the course can not be found in this book. We will distribute complementary lecture notes and reading materials along the course for these topics. We also recommend the students to read the book Campbell, Reece and Simon, Essencial Biology, for a general idea of biology.
Assignments: We will have 4 take-home assignments.
Grading: One mid-term exam (30%), Combined assignments (30%), Final exam (40%).
Office hour: Tuesday and Thursday (11:00am-noon), or upon appointment.
Prerequisites: No particular knowledge required, except high school level chemistry/biology and most important common sense .
Introduction to Peptide Science
Peptides are biomolecules comprised of amino acids which play an important role in modulating many physiological processes in our body. This book presents an interdisciplinary approach and general introduction to peptide science, covering contemporary topics including their applicability in therapeutics, peptide hormones, amyloid structures, self-assembled structures, hydrogels, and peptide conjugates including lipopeptides and polymer-peptide conjugates. In addition, it discusses basic properties and synthesis clearly and concisely.
Taking a logical approach to the subject, Introduction to Peptide Science gives readers the fundamental knowledge that is required to understand the cutting-edge material which comes later in the book. It offers readers in-depth chapter coverage of the basic properties of peptides synthesis amyloid and peptide aggregate structures antimicrobial peptides and cell-penetrating peptides and peptide therapeutics and peptide hormones.
- Introduces readers to peptide science, including synthesis and properties
- Provides unique content covering properties, synthesis, self-assembly, aggregation, and applications
- Summarizes contemporary topics in an accessible fashion including applications in therapeutics, peptide hormones, amyloid structures, self-assembled structures, hydrogels, and peptide conjugates including lipopeptides
- Presented at an introductory level for the benefit of students and researchers who are new to the subject
Introduction to Peptide Science is an ideal text for undergraduate students of chemistry, biochemistry, and other related biological subjects, and will be a valuable resource for postgraduate students and researchers involved in peptide science and its applications.
AP Sample 4 Lab 2 – Enzyme Catalysis
This lab will observe the conversion of hydrogen peroxide to water and oxygen gas by the enzyme catalysis. The amount of oxygen generated will be measured and used to calculate the rate of the enzyme-catalized reaction. Enzymes are proteins produced by living cells. Enzymes act as biochemical catalysts during a reaction, meaning they lower the activation energy needed for that reaction to occur. Through enzyme activity, cells gain the ability to carry out complex chemical activities at relatively low temperatures. The substance in an enzyme-catalyzed reaction that is to be acted upon is the substrate, which binds reversibly to the active site of the enzyme. The active site is the portion of the enzyme that interacts with the substrate. One result of this temporary union between the substrate and the active site is a reduction in the activation energy required to start the reaction of the substrate molecule so that products are formed. In a mathematical equation of the substrate (S) binding with the activation site (E) and forming products (P) is:
Several ways enzyme action may be affected include:
1) Salt Concentration — For example, if salt concentration is close to zero, the charged amino acid side chains of the enzyme molecules will attract each other. The enzyme will then denature and form an inactive precipitate. If salt concentration is extremely high, the normal interaction of charged groups will be blocked, new interactions will occur, and again the enzyme will precipitate. An intermediate salt concentration such as that of human blood (0.9%) is the optimum for many enzymes.
2) pH of the environment — . The pH of a solution is a logarithmic scale that measures the acidity or H+ concentration in a solution. The scale begins at 0, being the highest in acidity, and ends at 14, containing the least amount of acidity. As the pH is lowered an enzyme will tend to gain H+ ions, disrupting the enzyme’s shape. In turn, if the pH is raised, the enzyme will lose H+ ions and eventually lose its active shape.
3) Temperature — Usually, chemical reactions speed up as the temperature is raised. When the temperature is increased, more of the reacting molecules have enough kinetic energy to undergo the reaction. However, if the temperature goes past a temperature optimum, the conformation of the enzyme molecules is disrupted.
4) Activations and Inhibitors — Many molecules other than the substrate may interact with an enzyme. If such a molecule speeds up the reaction it is an activator, but if it slows the reaction down it is an inhibitor.
The enzyme used in this lab is catalase, which has four polypeptide chains that are composed of more than 500 amino acids each. One function of this enzyme is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes. Catalase is also involved in some of the many oxidation reactions that occur in the cells of all living things. The primary reaction catalyzed by catalase is the decomposition of hydrogen peroxide to form water and oxygen.
Without the help of catalase, this reaction occurs spontaneously, but very slowly. Catalase helps to speed up the reaction considerably. In this lab, a rate for this reaction will be determined.
The enzyme catalase, under optimum salt conditions, temperature, and pH level will speed up the reaction as it denatures the hydrogen peroxide at a higher rate than normal.
For the first part of the lab, 10 mL of 1.5% H2O2, a 50-mL glass beaker, and 1 mL of fresh catalase are needed. At the second stage a test tube, a hot water bath, 5 mL of catalase, 10 mL of 1.5% H2O2 are needed. Finally, in the third part, a potato, and10 mL of 1.5% H2O2 are needed.
For this experiment, 10 mL of 1.5% H2O2, 1 mL of water, 10 mL of sulfuric acid, two 25 mL beakers, 5-10 mL syringe, potassium permanganate, lab aprons and trays are needed.
In this section of the experiment, 20 mL of 1.5% H2O2, two glass beakers, 1 mL of H2O, 10 mL of sulfuric acid, a 5 mL syringe, 5-10 mL of potassium permanganate, and lab aprons and trays are used.
In the final part of the lab, 6 plastic cups labeled 10, 30, 60, 120, 180, 360, 6 plastic cups labeled acid, 60 mL of 1.5% H2O2, a 50-mL beaker, 6 mL of catalase extract, two 5-mL syringes, potassium permanganate, a timer (clock), lab aprons and trays are needed.
Transfer 10 mL of 1.5% H2O2 into a 50-mL glass beaker and add 1 mL of freshly made catalase solution. The fresh catalase should be kept on ice until ready to be used. Observe the reaction. Then transfer 5 mL of purified catalase extract to a test tube and place it in a hot water bath for five minutes. Transfer 10 mL of 1.5% H2O2 into a 50-mL beaker and add 1 mL of the boiled catalase solution, after it has cooled. Observe the changes in the reaction. To demonstrate the presence of catalase in living tissue, cut 1 cubic cm of potato, macerate it, and transfer it into a 50-mL glass beaker containing 10 mL of 1.5% H2O2. Observe the results.
Put 10 ml of 1.5% H2O2 into a clean glass beaker. Add 1 mL of H2O. Add 10 mL of sulfuric acid (1.0 M). USE EXTREME CARE IN HANDLING ACIDS. Mix the solution well. Remove a 5 mL sample. Place this 5 mL sample in another beaker, and assay for the amount of H2O2 as follows: Place the beaker containing the sample over white paper. Use a burette or 5 mL pipette to add potassium permanganate a drop at a time to the solution until a persistent pink or brown color is obtained. Remember to gently swirl the solution after adding each drop.
To determine the rate of spontaneous conversion of H2O2 to H2O and O2in an uncatalyzed reaction, put about 20 mL of 1.5% H2O2 in a beaker. Store it uncovered at room temperature for approximately 24 hours. Put 10 mL of 1.5% H2O2 into a clean glass beaker (using the uncatalyzed H2O2 that set out). Add 1 mL of H2O2 and then add 10 mL of sulfuric acid (1.0 M). Be careful when using acid. Mix this solution well. Remove a 5 mL sample and place it into another beaker. Assay for the amount of H2O2 as follows: Use a 5 mL syringe to add one drop of potassium permanganate at a time to the solution until it becomes a persistent pink or brown color. Gently swirl the solution after adding each drop. Record all results.
If a day or more has passed since Exercise B was performed, a baseline must be reestablish. Repeat the assay from Exercise B and record the results. Compare with other groups to check that results are similar. To determine the course of an enzymatic reaction, how much substrate is disappearing over time must be measured. The first thing to be done is to set up the six cups labeled with times, and the other six, one directly in front of each cup with a time on it. Then put 10 mL of 1.5% H2O2 into the cup marked 10 sec. Add 1 mL of catalase extract to this cup. Swirl gently for 10 seconds using a timer or clock for help. At 10 seconds, add 10 mL of sulfuric acid. Remove 5 mL and place in the cup directly in front of the cup marked 10 sec. Assay the 5 mL sample by adding one drop of potassium permanganate at a time until the solution turns a pink or brown. Repeat the previous steps, with clean cups using the times 30, 60, 120, 180, and 360. Record all results and observations.
AP Lab 2 Report 2001
Enzymes are proteins produced by living cells that act as catalysts, which affect the rate of a biochemical reaction. They allow these complex biochemical reactions to occur at a relatively low temperature and with less energy usage.
In enzyme-catalyzed reactions, a substrate, the substance to be acted upon, binds to the active site on an enzyme to form the desired product. Each active site on the enzyme is unique to the substrate it will bind with causing each to have an individual three-dimensional structure. This reaction is reversible and is shown as following:
Enzymes are recyclable and unchanged during the reaction. The active site is the only part of the enzyme that reacts with the substrate. However, its unique protein structure under certain circumstances can easily be denatured. Some of the factors that affect enzyme reactions are salt concentration, pH, temperature, substrate and product concentration, and activators and inhibitors.
Enzymes require a very specific environment to be affective. Salt concentration must be in an intermediate concentration. If the salt concentration is too low, the enzyme side chains will attract each other and form an inactive precipitate. Likewise, if the salt concentration is too high, the enzyme reaction is blocked by the salt ions. The optimum pH for an enzyme-catalyzed reaction is neutral (7 on the pH scale). If the pH rises and becomes basic, the enzyme begins losing its H+ ions, and if it becomes too acidic, the enzyme gains H+ ions. Both of these conditions denature the enzyme and cause its active site to change shape.
Enzymes also have a temperature optimum, which is obtained when the enzyme is working at its fastest, and if raised any further, the enzyme would denature. For substrate and product concentrations, enzymes follow the law of mass action, which says that the direction of a reaction is directly dependent on the concentration. Activators make active sites better fit a substrate causing the reaction rate to increase. Inhibitors bind with the enzymes’ active site and block the substrate from bonding causing the reaction to subside.
The enzyme in this lab is catalase, which produced by living organisms to prevent the accumulation of toxic hydrogen peroxide. Hydrogen peroxide decomposes to form water and oxygen as in the following equation:
This reaction occurs spontaneously without catalase, but the enzyme speeds the reaction considerably. This lab’s purpose is to prove that catalase does speed the decomposition of hydrogen peroxide and to determine the rate of this reaction.
The enzyme catalase, under optimum conditions, effectively speeds the decomposition of hydrogen peroxide.
Exercise 2A: Test of Catalase Activity
In Part 1, the materials used were 10mL of 1.5% H2O2, 50-mL glass beaker, 1 mL catalase, and 2 10-mL pipettes and pipette pumps. In Part 2, the materials used were 5 mL of catalase, a boiling water bath, 1 test tube, a test tube rack, 10 mL of 1.5% H2O2, 50-mL beaker, and 2 10-mL pipettes and pipette pumps. In Part 3, the materials used were 10 mL of 1.5% H2O2, 50-mL beaker, liver, and a syringe.
Exercise 2B: The Baseline Assay
This part of the lab required 10 mL of 1.5% H2O2, 1 mL distilled H2O, 10 mL of H2SO4, 2 50-mL beakers, a sheet of white paper, 5 mL KMnO4, 2 5-mL syringes, and 2 10-mL pipettes and pumps.
Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition
The materials used for this section were 15 mL of 1.5% H2O2, 1 mL distilled H2O, 10 mL H2SO4, 2 50-mL beakers, a sheet of white paper, 5 mL KMnO4, 2 5-mL syringes, and 2 10-mL pipettes and pumps.
Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition
The materials required for Exercise 2D were 70 mL of 1.5% H2O2, 70 mL of H2SO4, 6 mL of catalase solution, 13 plastic, labeled cups, 3 100-mL beakers, 1 50-mL beaker, 1 10-mL syringe, 1 5-mL syringe, 1 60-mL syringe, a sheet of white paper, a timer, and 30 mL of KMnO4.
Exercise 2A: Test of Catalase Activity
In Part 1, 10 mL of 1.5% H2O2 were transferred into a 50-mL beaker. Then, 1 mL of fresh catalase solution was added and the reaction was observed and recorded. In Part 2, 5 mL of catalase was placed in a test tube and put in a boiling water bath for five minutes. 10 mL of 1.5% H2O2 were transferred to a 50-mL beaker and 1 mL of the boiled catalase was added. The reaction was observed and recorded. In Part 3, 10mL of 1.5% H2O2 were transferred to a 50 mL beaker. 1 cm3 of liver was added to the beaker and the reaction was observed and recorded.
Exercise 2B: The Baseline Assay
10 mL of 1.5% H2O2 were transferred to a 50-mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded. The baseline assay was calculated.
Exercise 2C: The Uncatalyzed Rate of H2O2 Decomposition
A small quantity of H2O2 was placed in a beaker and stored uncovered for approximately 24 hours. To determine the amount of H2O2 remaining, 10 mL of 1.5% H2O2 were transferred to a 50-mL beaker. 1 mL of H2O was added instead of catalase, and then, 10 mL of H2SO4 were added. After mixing well, a 5 mL sample was removed and placed over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded. The percent of the spontaneously decomposed H2O2 was calculated.
Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition
The baseline assay was reestablished following the directions of Exercise 2B. Before starting the actual experiment a lot of preparation was required. Six labeled cups were set out according to their times and 10 mL of H2O2 were added to each cup. 6 mL of catalase were placed in a 10-mL syringe, and 60 mL of H2SO4 were placed in a 60-mL syringe. To start the actual lab, 1 mL of catalase was added to each of the cups, while simultaneously, the timer was started. Each of the cups were swirled. At 10 seconds, 10 mL of H2SO4 were added to stop the reaction. The same steps were repeated for the 30, 60, 120, 180, and 360 second cups, respectively.
Afterwards, a five 5 mL sample of each of the larger cups were moved to the corresponding labeled smaller cups. Each sample was assayed separately by placing each over a white sheet of paper. A 5-mL syringe was used to add KMnO4, 1 drop at a time until a persistent brown or pink color was obtained. The solution was swirled after every drop, and the results were observed and recorded.