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# 2008 University of South Africa All rights reserved Printed and published by the University of South Africa Muckleneuk, Pretoria BCH3711/1/2009^2011 98292870 3B2
Learning unit Welcome! 1 ENZYME NOMENCLATURE AND THE STRUCTURE AND FUNCTION OF ENZYMES 1 2 3 4 5 6 7 8 9 2 Time Textbook reference Learning outcomes Background to learning unit Introduction to enzymology The structure of protein Specifities of enzyme functioning Monomeric and oligomeric enzymes Self-evaluation Page (v) 1 1 1 1 1 2 2 2 3 3 4 4 4 4 4 4 5 5 5 5 6 6 6 6 6 7 7 7 9 9 9 9 9 10 10 11
BIOENERGETICS, CATALYSIS AND KINETICS 1 2 3 4 5 6 7 8 9 Time Textbook reference Learning outcomes Background to learning unit General concepts in bioenergetics Factors affecting the rate of chemical reactions The kinetics of uncatalysed and enzyme-catalysed reactions The nature of enzyme-catalysed reactions Self-evaluation
KINETICS OF SINGLE-SUBSTRATE ENZYME-CATALYSED REACTIONS 1 2 3 4 5 6 7 Time Textbook reference Learning outcomes Background to learning unit The relationship between vo and [So] Rapid-reaction kinetics and relaxation kinetics Self-evaluation and additional sources
ENZYME INHIBITION 1 2 3 4 5 6 7 Time Textbook reference Learning outcomes Background to learning unit Reversible inhibitors Irreversible inhibitors Self-evaluation and additional sources
(iv) Learning unit 5 KINETICS OF MULTISUBSTRATE ENZYME-CATALYSED REACTIONS 1 2 3 4 5 6 7 8 6 Time Textbook reference Learning outcomes Background to learning unit Reaction mechanisms of multisubstrate enzyme-catalysed reactions Steady-state condition kinetics Experimental approaches to the investigation of multisubstrate enzymecatalysed reactions Self-evaluation and additional sources Page 12 12 12 12 12 13 13 13 13 15 15 15 15 15 16 16 16 16 18 18 18 18 18 19 19 19 20 20 THE ACTIVE CENTRE AND THE CHEMICAL NATURE OF ENZYME CATALYSIS 1 2 3 4 5 6 7 8 Time Textbook reference Learning outcomes Background to learning unit Identifying the active site of enzymes Mechanisms of catalysis The use of co-factors in catalysis Self-evaluation 7 ALLOSTERIC ENZYMES AND THE SIGNIFICANCE OF SIGMOIDAL KINETICS 1 2 3 4 5 6 7 8 9 Time Textbook reference Learning outcomes Background to learning unit Binding of ligands to proteins Mechanisms of catalysis Sigmoidal kinetics and allosteric enzymes The significance of sigmoidal behaviour Self-evaluation .
Tutorial letters and study guides If you have received no tutorial letters or study guides. should be sent directly to the Registrar. For telephonic enquiries. No tutorial matter can be obtained from the Department of Life Sciences in Potchefstroom. as it largely forms the basis of your grasp of biochemistry as a whole. We trust that you will find this introduction to enzymology not only instructive and interesting. These topics have great relevance for you as a student of natural sciences. please contact the Department of Despatch at the main Unisa campus in Pretoria. will stand you in good stead here. that correspondence addressed to different departments should be placed in different envelopes. Unisa. The focus points of this course are general principles of enzyme functioning. such as enquiries about changes of address and phone numbers. especially protein chemistry. This course also supports your other third year biochemistry modules ö metabolism and molecular biology ö as enzymes are the catalysts in all biochemical processes. Correspondence addressed to the same department may be placed in one envelope. It is very important to write your student number. Administrative enquiries All administrative enquiries. . however. that you gained in your second year modules. Written enquiries should be clearly marked for the attention of the Department of Despatch. Please note. The knowledge of basic biochemical concepts.We are excited about introducing you to this subject. All telephonic administra- .Our purpose is also to demonstate to you how the properties of enzymes kan be used for various applications in natural science and biotechnology (BCH3714). subject and course code clearly in the top right hand corner of all correspondence.(v) BCH3711/1 Welcome! Welcome to this module! This is your second introduction to a specific subsection of biochemistry: the study of enzymes or enzymology. each marked clearly for the attention of the department involved. enzyme kinetics and the role of enzymes in metabolism. use only the telephone number given on the inventory letter. Correspondence Address all correspondence to: The Registrar (Academic) PO Box 392 UNISA 0003 . but also highly enjoyable! . as it will give you insight into the nature and role of enzymes in living systems.
Please note that practical work and theory in Biochemistry are an entity. P & Palmer. structures. et cetera. istry. EVALUATION Examination One two hour paper will be written in November. ADMISSION TO THE EXAMINATION Admission to the examination will be granted only if you have obtained a minimum of 100 credits for assignments during the year. You have to obtain 50% to pass the examination. T 2007 Enzymes: Biochemistry. drawings. are concretely handled. West Sussex. Tutorial Letter 101 for BCH3111 contains the assignments and information about the closing dates and credits. tion. 2nd edition. England: Hor wood. et cetera ö keep accurate records of the practical work you have . literature search facilities and interactive websites. concepts. systems. but the second edition may be the only one available. 1st edi. The date.We would like to encourage you to gain internet access for this module. ISBN 1904275273. Clinical Chem.West Sussex. and as such contribute to the attainment of outcomes.) Bonner. Biotechnology. STUDY MATERIAL The textbook for this module is: (The contents of the first and second edition are exactly the same. time and examination centre are indicated in Part 1. T 2001.(vi) tive enquiries should be made to the relevant sections. and you will receive written confirmation in September. certain aspects of this module forms part of the practical module BCH3714. It is very important. results. that you work through the program systematically and ö in the the form of reports. Section C of the Unisa Calender. However. During the practicals theory becomes reality when specimens. This includes databases. which you received with your study material at registration. OR Palmer. Consult the booklet Unisa: services and procedures. Chichester. processes. ISBN 1898563780. as it can be a good source of information. Clinical Chemistry. Additional sources (books) as well as a variety of internet sources are recommended in the different learning units. and no practical work will be done in this module. Chichester. ideas. Please note that the information in the study guide is based on the page numbers of the first edition. Enzymes: Biochemistry. graphs. therefore. England: Horwood. . Biotechnology. PRACTICALS AND PRACTICAL EXAMINATIONS BCH3711 is a theory module.
. concise and authoritative description of a concept to demonstrate its meaning in no uncertain terms. . . which involves a clear. A sound theoretical background is essential for success in BCH3714 . always contain certain key or action words. To assist you in this. Do not describe or discuss one matter fully before you move on to the next. Facts. Make it your goal to ensure that you attain all the outcomes formulated at the start. . . indicating similarities and differences.(vii) BCH3711/1 done. incidents or problems should be contrasted throughout. which will be presented at the Potchefstroom campus of the Northwest University in September. and give reasons for statements and results. Compare You have to be careful here. Make use of illustrations. facts or results in a logical. whether in assignments or examinations. Define Reproduction of knowledge is required. . ACTION WORDS Questions. Provide an overview An extensive volume of knowledge should be summarised logically and systematically and conveyed without losing sight of the essence of the matter. Prove Substantiate the facts by logically presenting acceptable facts. . Explain Formulate the concept as simply as possible to ensure that the reader understands it.You should know what these action words mean and what is required when you answer the questions. Name Just write down the bare facts as briefly as possible. descriptions and examples. well-structured way. a short list of these words is supplied below. . No commentary or discussion is necessary. Describe Here we require you to demonstrate your knowledge and to relate properties.
catalysis and kinetics Kinetics of single-substrate enzyme-catalysed reactions Enzyme inhibition Kinetics of multisubstrate enzyme-catalysed reactions The active centre and chemical nature of enzyme catalysis Allosteric enzymes and the meaning of sigmoidal kinetics . procedures and techniques ö cope with concrete and abstract problems by using supporting evidence and theory-driven arguments ö develop the abililty to obtain subject-directed information and do critical analyses and syntheses of quantitative and/or qualitative data . principles and theories. GENERAL OUTCOMES The South African Qualifications Authority Act 58 of 1995 requires university students to achieve the following outcomes at third year level: . This requires you to: ö function in a fluctuating and unfamiliar learning environment by using your initiative and being responsible ö evaluate yourself. This requires you to ö obtain a well-rounded knowledge of this module ö obtain a coherent and critical understanding of this subject and its terms.(viii) . regulations. astutely and correctly MODULE PLAN This module is divided into the following seven learning units: Learning unit 1 Learning unit 2 Learning unit 3 Learning unit 4 Learning unit 5 Learning unit 6 Learning unit 7 Enzyme nomenclature and the structure and function of enzymes Bioenergetics. Causes and results are detected and relationships determined. Discuss Discussion presupposes insight and discernment when it comes to application and judgement. . Here we expect you to analytically investigate and discuss different aspects of the matter or statement. and be able to place your knowledge and understanding within the broader framework of biological and other knowledge ö know the basic applications. and your ability to address your learning needs. . Competence. Analyse The contents are divided into parts or elements and discussed. Evaluate Here we expect you to judge something on the basis of certain criteria and express a well-motivated value judgement. Autonomous learning.
enzyme inhibition and the different mechanisms .(ix) MODULE OUTCOMES By the end of this module. data processing and data interpretation . the concepts of catalysis and kinetics of single. the sigmoidal behaviour of enzymes and their importance with respect to metabolic regulation . the nomenclature of enzymes . the properties of allosteric enzymes.and multi-substrate enzymecatalysed reactions . you will be conversant with BCH3711/1 . experimental approaches to enzyme kinetics. the determination and meaning of enzyme-kinetic parameters .
1 BCH3711/1 Learning unit 1 ENZYME NOMENCLATURE AND THE STRUCTURE AND FUNCTION OF ENZYMES 1 TIME You need about 10 hours to complete this learning unit successfully. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to . give an historical overview of enzymes . describe and identify the EC (Enzyme Commission) classification of enzymes by means of examples . Apart from their role as catalysts in all biological processes. although enzyme preparations have been used by humans in processes such as fermentation for millenia. demonstrate a well-rounded and systematic knowledge of the structural aspects of enzymes and protein . Chapters 1±5. Clinical Chemistry (2nd edition). discuss and compare the relationship between the structure and functions of monomeric and oligomeric enzymes with reference to relevant examples 4 BACKGROUND TO LEARNING UNIT Enzymes were identified as biological catalysts as early as the 19th century. Biotechnology. enzymes have become indispensable to the biomedical profession and in- . 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistr y.
1 Study the descriptions of enzymes in chapter 1. In this learning unit we will give an overview of the history of the study of enzymes and general terminology.2 You should be able to provide an overview of the history of the study of enzymes (pp 3^4). . effect of salts. . 5 INTRODUCTION TO ENZYMOLOGY 5. . You should be able to distinguish clearly between the six classes of enzymes according to naming.4 You should know the meaning of the terms ``salting in''.The first enzymes were described early in the 20th century. numbering and type of reaction they catalyse ö we expect you to be thorough and give evidence of insight into the system. Use the textbook to refresh your memory (pp 16^56). 5. 6 THE STRUCTURE OF PROTEIN 6. 5. and in the course of a relatively short period (about one century) a lot has happened: enzymes taken from natural sources were purified and utilised. This was fully dealt with in your second year.11 (pp 61^64).You should be able to classify an enzyme reaction in the group to which it belongs.2 You should be able to describe the components and general properties of the active centre.3 You can study an outline of the acid-based properties of protein (pp 56^57). so we expect you to have a sound and systematic knowledge of this topic.1 You should be able to distinguish between the type specifities of enzymes (p 67).You should also know the biochemical principles underlying these processes. .12 and Figure 3.2 In order to understand the functioning of enzymes. The examples given in the textbook should be studied (pp 4^12). ``ionic strength'' ``insoluble complexes'' ``denaturation'' and ``isoelectric point'' and the . 6. 6. page 3. 7 SPECIFITIES OF ENZYME FUNCTIONING 7. and should understand the numbering system of the IUBMB's Enzyme Commission. .2 dustry. you should have a general knowledge of the structure and properties of amino acids and the levels of protein structure. ``salting out''. pH and temperature. 6. . and the use of recombinant enzymes has become commonplace in medicine and industry. It is important that you know what is meant by conjugated proteins and that you are able to give examples of these (p 15). ``coenzyme'' and ``prosthetic group'' to enable you to give a description of each and draw comparisons. Please pay particular attention to Figure 3. You should have insight into the impact of changes in external .1 A basic knowledge of protein structure is vital in enzymology. 7. . as well as the meanings of the terms ``substrate'' ``product'' ``co-factor'' ``apoenzyme'' ``holoenzyme'' .3 The classification of enzymes according to the system of the IUBMB's Enzyme Commission is of importance here.
3 Visit the website of the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) at http://www. temperature and ion strength upon these components (pp 68^70).chem.qmw.1 and 1. pp 70^73). .2 To test your general knowledge of protein structure after studying it. 9. write a summary of about 20 lines about the structure of protein. 9. After solving these problems. but will be of great help if you wish to become acquainted with international conventions regarding enzyme terminology (general terms that have been agreed upon). you should be able to describe the different hypotheses regarding enzyme functioning and discuss the respective aspects that are addressed by these hypotheses and those that are not addressed (Fig 4. find the answers at the back of your textbook and compare them with your answers.1 Problems 1.uk/ iubmb/kinetics/ and compare the terminology and classification of enzymes compiled by this commission in 1981 with those in your textbook.3 With reference to the components of the active centre studied in 7.1 You should be able to distinguish between monomeric and oligomeric enzymes with respect to structure and by means of relevant examples (pp 76^84). Solve these problems as far as possible (the textbook contains limited information on the classification of the 2nd to 4th EC number of the different classes) to ensure that you have achieved the second outcome of this learning unit. Do the same exercise after studying chapter 4. 8 MONOMERIC AND OLIGOMERIC ENZYMES 8.2 on pages 12 to 14 are focussed on the classification of enzymes. Now compare it with the summary at the end of chapter 3 (p 64).ac.2. 9 SELF-EVALUATION 9. This will help you to evaluate your knowledge of and insight into the third and fourth learning outcomes. This is not compulsory.3 BCH3711/1 factors such as pH. 7.2^4.
Biotechnology. energy and reaction rate. 2nd edition. It also forms the basis of the next three learning units. 5 GENERAL CONCEPTS IN BIOENERGETICS 5. describe the general concepts of bioenergetics and explain the meaning of each concept by citing examples in biochemistry 4 BACKGROUND TO LEARNING UNIT This learning unit focusses on the principles of physics and chemistry used in enzyme kinetics.4 Learning unit 2 BIOENERGETICS. 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistr y. This includes the laws of thermodynamics. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to . It also serves as critically important background knowledge for the next learning units. CATALYSIS AND KINETICS 1 TIME You need about 16 hours to complete this learning unit successfully. and outlines the background and principles underlying enzyme kinetics. Clinical Chemistry.1 You should gain an understanding of and insight into the following concepts: . Chapter 6. where enzyme kinetics will be investigated.
1 You should be able to discuss the kinetics of uncatalysed reactions. 7. and should understand the Principle of Mass Action and the concept of reaction order.1^2.1 Take note of the methods used to investigate the kinetics of enzyme-catalysed reactions. and be able to describe them. 8 THE NATURE OF ENZYME-CATALYSED REACTIONS 8.1 For self-evaluation. is suited to the role it fulfils (pp 90^91).1 and 6. free energy (DG) and standard free energy of change (DGK) (pp 87^90). (Fig 6.2 You should be able to describe and explain the course followed by the freeenergy profile of an enzyme-catalysed reaction in which a substrate and enzyme product complex is formed. 7 THE KINETICS OF UNCATALYSED AND ENZYME-CATALYSED REACTIONS 7.1 You should know the three factors that affect the rate of chemical reactions. You should be able to give the reaction rate equation (v) for first and second order reactions and to make graphic representations of the initial reaction rate as against reactant concentration (pp 96^98). which is based on it. 5. do problems 6. enthropy (DS). 7. and to give reaction equations for vo and Vmax (pp 99^100). 6 FACTORS AFFECTING THE RATE OF CHEMICAL REACTIONS 6.2 Get an overview of the historical introduction to the kinetics of enzyme-catalysed reactions (p 98). pp 91^95). Pay special attention to initial reaction studies. enthalpy (DH). as they will be used later in this module (pp 100^104). Make sure that you can distinguish between the terms ``collision theory'' ``activation energy. 9 SELF-EVALUATION 9.5 BCH3711/1 the First and Second Laws of Thermodynamics.3 You should be able to describe the relation between vo and [So ] at constants [Eo ] and the order in which the reaction takes place.2 on page 105 in the textbook.1) and ``transitional state'' .'' (Fig 6. .2 You should be able to describe the principle of common intermediates and the role as well as the structural properties of ATP and to explain why this molecule . 8. The answers are given at the back of your textbook.
and identify the assumptions on which this relationship is based . describe the concepts ``pre-steady-state kinetics'' and ``relaxation kinetics'' 4 BACKGROUND TO LEARNING UNIT In learning unit 1 we saw that enzyme reactions can be described according to substrate specificity. Biotechnology. 2nd edition. describe the principles underlying steady-state kinetics as presented by Briggs and Haldane. and derive the Michaelis-Menten equation . Clinical Chemistry. Certain kinetic paramaters are used here. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to . 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistr y. it is also necessary to describe how rapidly and effectively an enzyme can work to bind its substrate and turn it into product. We also examine the assump- . determine and explain the meaning of the most common enzyme-kinetic parameters by means of problems . Chapter 7. However. describe the relationship between initial reaction rate and substrate concentration as defined by Henri. and in this learning unit we investigate the relatively simple kinetics of single-substrate reactions.6 Learning unit 3 KINETICS OF SINGLE-SUBSTRATE ENZYME-CATALYSED REACTIONS 1 TIME You need about 16 hours to complete this learning unit successfully. Michaelis and Menten.
Eadie-Hofstee. You should also be able to fully describe the deductions made from them. Kcat and Kcat/Km. and to solve problems in which these parameters are used (pp 111^113).You should also be able to identify the differences between the two approaches. 5 THE RELATIONSHIP BETWEEN vo AND [SO] 5.You will also be introduced to the way in which the most common kinetic parameters such as vo.4 The use of primary graphic methods to determine the values of Km and Vmax is very important. Km. Note the possible advantages and disadvantages of each of the three methods. 6. and to describe and apply the adaptation of the equation for vo (pp 109^111). and indicate the relationship between vo and [So ] (Michaelis-Menten equation) (pp 107^109).7 BCH3711/1 tions we have to make in order to describe the vitally important relationship between initial reaction rate vo and substrate concentration [So ]. but it's a . We confine ourselves to three approaches: those of Lineweaver-Burk.2 You should be able to present the theoretical course of single substrate reactions and to state the meaning of the terms ``induction period''. which were derived from an equilibrium state view of a single substrate reaction. and Hanes. The terminology used in your textbook will suffice.3 You should be able to give an overview or the meaning of relaxation kinetics and its applications (pp 122^123). Km and Kcat are calculated. and be able to apply the graphic representation in order to calculate Km and Vmax. ``pre^steady state'' and ``steady state'' in their respective contexts (pp 120^121).1 Steady-state kinetics is based on the six commonly used assumptions as defined by Henri. Michaelis and Menten. and you will learn what these parameters mean.1 Get an overview of the use of rapid-reaction kinetics as well its concomitant measuring techniques. Vmax. 7 SELF-EVALUATION AND ADDITIONAL SOURCES 7.3 You should be able to fully describe the meaning. and be aware that the calculation of these parameters can also be done directly from the equations for vo (ie the Michaelis-Menten equation) (pp 113^117). 5. Ks. 5. 5. namely to adopt a steady-state approach to the enzyme-substrate complex. These assumptions are important. and take note of the limitations of thermodynamics in the study of metabolism (pp 118^110). You should be able to identify and describe these assumptions. as they are of special value when it comes to experimental investigation. 6.1 The terms used in enzyme kinetics sometimes differs from those that are commonly used. In each of these cases you should know which modification of the Michaelis-Menten equation was carried out. 6 RAPID-REACTION KINETICS AND RELAXATION KINETICS 6.2 You should be able to describe the adaptations of the Michaelis-Menton deductions suggested by Briggs and Haldane. units and calculation of the parameters Vmax.
2nd edition.1974.2 graphically before you can proceed to the next step when solving this problem.3 If you have access to a library. Engel. Enzyme kinetics: the steady-state approach. DL (ed). Christensen. . but please note that you should do the calculation of vo in problem 7. Contemporary enzyme kinetics and mechanism. Enzyme kinetics: a learning program for students of the biological and medical sciences. PC. GA. New York: Academic Press. do problems 7. 1983. 1981.chem.2 For self-evaluation. The answers are given at the back of the textbook. London: Chapman & Hall. ISBN 0412239701. ISBN 0721625916. 2nd edition.uk/iubmb/kinetics/.1 and 7.qmw.ac. ISBN 0125680503. NB: always use graph paper! 7. Purich.8 good idea to acquaint yourself with the conventions regarding notation in enzymology by visiting the website of the IUBMB at http://www. . 7. . HN & Palmer. you can consult the following sources to supplement your reading or to do extra enzyme kinetics problems for self-evaluation (not compulsory): .2 on page 127 in the textbook. London: WB Saunders.
recognise the role of enzyme inhibitors in metabolism . 2nd edition.9 BCH3711/1 Learning unit 4 ENZYME INHIBITION 1 TIME You need about 12 hours to complete this learning unit successfully. several pathological conditions. Enzymes were created in such a way that they can be regulated by particular inhibitors. to describe and execute the methods used when investigating the identification and kinetics of enzyme inhibitors. inhibitors ö especially irreversible inhibitors ö can stop certain reactions. which can lead to cell . On the other hand. transcription or translation process. are a classical example of this. Antibiotics. If biological reactions are not regulated. Chapter 8. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to . which usually act as inhibitors of a bacterial replication. Enzyme inhibitors are also designed and used in the pharmaceutical industry to suppress certain processes. can develop (deficiency in the regulation of lycil oxidase by Vit C).Biotechnology. such as scurvy. with reference to practical examples 4 BACKGROUND TO LEARNING UNIT The inhibition of enzyme reactions is a normal biological process and a critically important regulating mechanism in metabolic reactions. describe the classification and functioning of reversible and irreversible enzyme inhibitors by citing examples . 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistry. Clinical Chemistry.
A problem-solving approach will be followed in the evaluation of your abilities.2. 5.1 You should be able to describe the nature of this type of inhibition by referring to examples.3 You should be able to describe the Lineweaver-Burk equation in the presence of the inhibitor. it is very important to thoroughly investigate this phenomenon.2. substrate and allosteric inhibition (all reversible inhibition mechanisms) in outline form (pp 148^149). do correct calculations and make motivated deductions about the nature and mechanism of enzyme inhibition. 5 REVERSIBLE INHIBITORS 5.2 You should also be able to accurately distinguish between the types of reversible inhibitors.1 The inhibition mechanism.3 You should be able to compare partial. 5. you should be able to explain when an inhibitor should be regarded as irreversible (pp 149^151). 6 IRREVERSIBLE INHIBITORS 6. 5. 5.10 death.5 You should have the cognitive skills to process experimental data correctly. and to describe or predict and explain the effect of the inhibitor on the Km and Vmax values. Several toxins work in this way.1 You should be able to accurately distinguish between the properties of reversible and irreversible inhibitors with reference to the examples in the textbook (p 129). Using the kinetic data. 5. 5. and give a short summary of its effect on Km and Vmax values. Use the following to guide you (pp 128^149): 5. and describe or predict the Lineweaver-Burke graph and the effect of the inhibitor on its course.2. .2).2. Therefore. and the structure of the inhibitor are important (Fig 8.2.4 You should also be able to calculate the inhibition constants Ki and KI from secondary graphs compiled from data obtained from the primary graph (Lineweaver-Burk graph). that is. you should be able to identify the type of inhibitor by means of the Lineweaver-Burk graph. where the inhibitor binds to the enzyme. In other words. and to calculate the inhibition constants Ki and KI by using the Michaelis-Menten equation.2 You should be able to describe the equilibrium-state kinetics of the different inhibitors by referring to the deviation caused to the MichaelisMenten equation.You should also be able to distinguish between reversible and irreversible inhibitors on the basis of binding properties and kinetic parameters. and to make a deduction as to the possible mechanism of an inhibitor on the basis of structural data regarding the inhibitor. and that can be harnessed by medical science and industry. You should be able to give an example of such an inhibitor. it is one of the aspects of enzymology that gives us more insight into metabolic regulation.
The answers are given at the back of the textbook. . 7. cyanide. do problems 8.3 Investigate and compare the mechanisms of the following inhibitors: DFP .11 7 SELF-EVALUATION AND ADDITIONAL SOURCES BCH3711/1 7.1 to 8.1 Pay the website of the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB) another visit at http:// www.qmw. These questions test your ability to achieve the third learning outcome of this learning unit.4 on pages 152 to 153. Please note that problem 8.uk/iubmb/kinetics/ and compare the classification and kinetics of inhibitors with those in your textbook.3 alludes to problem 7.This is not compulsory. and ethylene glycol.chem.2 For self-evaluation. You can also consult the additional sources listed in the previous learning unit. but will help you to obtain a proper overview of the classification of enzyme inhibitors. 7.ac.2 in the previous learning unit.
describe and distinguish the different multisubstrate enzymecatalysed reaction mechanisms . and one needs to examine the experimental approach to these mechanisms. which in kinetics is seen as an additional substrate. and demonstrate that you know how to apply these approaches by processing experimental data and making deductions about the different mechanisms from the data at your disposal 4 BACKGROUND TO LEARNING UNIT Most enzyme reactions use more than one substrate or a co-factor. . the kinetics is more complex. describe and explain the use of steady state kinetics and product inhibitors in the experimental investigation of multisubstrate enzyme-catalysed reaction mechanisms. Chapter 9. Therefore it is necessary to investigate and describe this type of reaction. 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistr y. 2nd edition. Also. Clinical Chemistry. classify.12 Learning unit 5 KINETICS OF MULTISUBSTRATE ENZYME-CATALYSED REACTIONS 1 TIME You need about 10 hours to complete this learning unit successfully. Biotechnology. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to .
3 You should be able to calculate kinetic parameters (Km.1 Make sure that you can accurately distinguish between the different types of sequential and nonsequential reaction mechanisms. Make use of the notation employed in the textbook (substrates AX and B with products A and BX in a transferase-type reaction) for . 6. an inhibitor. You should be able to describe and explain the theoretical basis of the use of primary graphs for this purpose. This is comparable to the work done in the case of Michaelis and Menten in learning unit 3. 6. You should also be able to point out the relation between a variable substrate. the reaction mechanism and the kinetic course a reaction follows. [Bo ] of [AXo ] and Alberty's equation for a ping-pong bi-bi-mechanism respectively (pp 157-159).and Vmax values) using Alberty's general rate equation.4 You should be able to describe Dalziel's general rate equation and the purpose it serves.1 To test your knowledge of multisubstrate enzyme-catalysed reaction mechanisms after studying them. Here you should be able to describe Alberty's general rate equation and the conditions under which this equation is valid (pp 157-158). In Figure 9. and to use secondary graphs to calculate Km values in two-substrate reactions (pp 159^160). draw a diagram in which you classify the types of mechanisms and reactions.1 Steady-state kinetics can be applied in the case of multisubstrate reactions. as well as the deductions made from these graphs. and be able to make deductions from experimental data in this regard.3 the differences seen here are clearly distinguished (pp 162^163). 7 EXPERIMENTAL APPROACHES TO THE INVESTIGATION OF MULTISUBSTRATE ENZYME-CATALYSED REACTIONS 7.2 You should also be able to indicate the possible uses of inhibitors by referring to Cleland's two rules and their significance. Dit is extremely important that you understand this table (p 166) well and are able to explain it (pp 163-167). Here you should make use of the enumerative table on page 66. Refer to the course of the reaction and the formation of intermediary complexes in each case.2 You should be able to describe the application of Alberty's general rate equation at high second substrate concentrations.1 The mechanism of a reaction is commonly determined by means of two approaches: the use of primary graphs and the use of inhibitors. 8 SELF-EVALUATION AND ADDITIONAL SOURCES 8.13 BCH3711/1 5 REACTION MECHANISMS OF MULTISUBSTRATE ENZYME-CATALYSED REACTIONS 5. 6 STEADY-STATE KINETICS 6. (ie steady-state kinetics) by means of primary graphs from experimental data (p 159). 6. 7. You should be able to identify a reaction mechanism on the basis of given data (pp 155^156).
8. Now compare your classification with the one in the textbook.qmw. It is of the utmost importance that you should be able to solve these problems.2 Also see the classification done by the IUBMB at http://www. This will help you to get used to the way in which notation of the reactions and classification of the mechanisms are done (first learning outcome). or consult the additional sources listed in learning unit 3. 8.chem. Please note that these problems make use of a combination of the two experimental approaches described in Section 7. Have a good look at the tabled data you received before making your selection. . The answers are given at the back of the textbook. do problems 9.3 For self-evaluation and to determine whether you have achieved the second learning outcome.14 each of the mechanisms.1.uk/ iubmb/kinetics/ if you have access to tye internet.1 and 9. Carefully note the graphic representations of your choice of variable substrates.2 on pages 170 to 171.ac.
Chapters 10 and 11. 2nd edition. 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistr y. mediate these interactions.We also examine the chemical nature of enzyme catalysis and the role . Biotechnology. describe the role of co-factors and co-enzymes in enzyme catalysis by means of examples from the metabolism 4 BACKGROUND TO LEARNING UNIT The interactions taking place in the active centre of enzymes determine the nature of the reaction and the kinetic course for that particular enzyme.15 BCH3711/1 Learning unit 6 THE ACTIVE CENTRE AND THE CHEMICAL NATURE OF ENZYME CATALYSIS 1 TIME You need about 5 hours to complete this learning unit successfully. give an onverview of the different catalytic mechanisms involved in enzymes. give a concise description of the different empirical methods used to investigate the active centre . In this learning unit we study the ways in which these interactions are investigated. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to . Certain amino acids in the catalytic sites. and describe two examples of catalytic mechanisms in enzymes . Clinical Chemistry. as well as co-factors that may be involved.
Possible internet sources are also listed below.1 This assignment will really help you to gain insight into the functioning of enzymes by means of concrete examples.You should be able to compare the following co-enzymes with regard to origins. Select any two (or more.1) Glutathione reductase (EC 22.214.171.124 of co-factors with reference to examples. use the information on these enzymes in domesticated bovine animals (Bos Taurus) or rats (Rattus Norvegicus).4 7 .2.1) Pyruvate dehydrogenase (EC 1.2.6 Nicotinamide nucleotides Flavine nucleotides Adenosine phosphates Co-enzyme A Biotin Co-enzyme B12 8 SELF-EVALUATION 8. if you like) of the following enzymes (or any other enzyme you're interested in!) in the list given below and try to track down the information (8.2.7) .1.2.21. covalent and enzyme catalysis.1. . . You can choose any organism. structure.2. electrostatic.6) on these enzymes. 5 IDENTIFYING THE ACTIVE SITE OF ENZYMES 5. In each case. and to apply this information to describe the catalytic mechanism of the enzymes chymotrypsin and lysozyme (pp 193^200).This knowledge will give us a better idea of the catalytic functioning of enzymes. and to achieve the outcomes of this learning unit.17) Hexokinase (EC 126.96.36.199.2 7 . . and the catalytic role they fulfil in enzyme reactions. but if you have doubts.1 7 . 7 THE USE OF CO-FACTORS IN CATALYSIS 7.1 You should be able to distinguish between the chemical processes of acidbased.1. Chymotrypsyn (EC 3.3 7 .2 Co-enzymes are used by several enzymes. make use of an example to illlustrate the technique (pp 175^187). 7.1) Lysozyme (EC 3.1^8.4.5 7 .1. although you can use other sources as well. . . 6 MECHANISMS OF CATALYSIS 6.1 You should be able to compare the properties and role of metal-activated enzymes with those of metallo-enzymes (pp 202^203). You should also be able to give one example of a situation in which enzyme reactions employ co-enzymes (pp 206^221): 7 .1.2.1 You should be able to give a concise description of the techniques commonly used to determine the substrate-binding and catalytic sites of an enzyme.
.brenda-enzymes.info/index. ions and inhibitors of enzyme Functional parameters (ie kinetic parameters.nih. .gov/sites/entrez?db=PubMed) Methods in Enzymology.nlm.5 8.1 8.1. .17 8.1.3 8.1.6 BCH3711/1 Systemic and common name Reaction catalysed and type Co-factors involved Catalytic mechanism and type.4 8.expasy.1.genome. .jp/kegg/) PUBMED (http://www.php4) EXPASY (http://au.ad.2 8.1. good sources include the following: . optimal pH and temperature. as well as active site of enzyme Metals.1. ISBN 0121821862 . pI) Apart from your textbook (which contains limited information).ncbi. Series by Academic Press. BRENDA (http://www.org/enzyme/) KEGG (http://www.
flow of genetic information. reproduce models describing sigmoidal kinetics and point out the limitations of these models . describe sigmoidal kinetics and co-operativity (different types) in enzymes. as well as the molecular interactions they involve . Enzymes fulfil an active role in this and have been constructed in such an extraordinary way that the regula- . Chapters 12. etc) is essential for the survival and functioning of the cell. demonstrate insight into the significance of sigmoidal kinetics with regard to enzymes in metabolic processes . Biotechnology. describe other metabolism regulation mechanisms and indicate the role of each mechanism in metabolism by citing examples 4 BACKGROUND TO LEARNING UNIT Control of all cellular processes (metabolic processes. 3 LEARNING OUTCOMES By the end of this learning unit you should be able to . 13. describe the principles of ligand binding to a protein with regard to binding constants and fractional saturation . 2 TEXTBOOK REFERENCE This learning unit is based on the textbook Enzymes: Biochemistr y. 2nd edition.18 Learning unit 7 ALLOSTERIC ENZYMES AND THE SIGNIFICANCE OF SIGMOIDAL KINETICS 1 TIME You need about 10 hours to complete this learning unit successfully. and 14. Clinical Chemistry.
and in this learning unit you will become better acquainted with these ways. negative. referring to the following main points (pp 241^246): . 7 SIGMOIDAL KINETICS AND ALLOSTERIC ENZYMES 7. 5. Distinguish between the socalled Tand R forms of a protein (pp 240^241). positive co-operativity and negative cooperativity.1 You should be able to define the binding constant Kb for a protein with a singleligand binding site. 6 MECHANISMS OF CATALYSIS 6. by means of a graph and making use of the relationship between Yand [S] (pp 229^232.19 BCH3711/1 tion of activities adapts itself to the environment and needs of the cell and organism. electrostatic. vo and [So ]. refer to the relationship between the binding constants Kb1 and Kb2 in your equation. 5. and to apply this information in describing the catalytic mechanism of the enzymes chymotrypsyn and lysozyme (pp 193^200).1 You should be able to define the term ``allosterism'' and describe the role of allosterism in the regulation of enzyme activities. 5.6 In order to illustrate the way in which co-operativity is typically investigated. 5. covalent and enzyme catalysis. 7.You should also be able to explain how one uses this equation in the Hill graph and indicate how deductions can be made from it (pp 225^228). and the Scatchard plots respectively can be used.2 You should be able to distinguish between positive. In each case. 5.4 a^d). 5 BINDING OF LIGANDS TO PROTEINS 5.You should also be able to describe the equation for Y in steady states (pp 223^224). Please make a special note of the conditions in which each of these three appraoches can be used (pp 233^236).1 You should be able to distinguish between the biochemical processes of acidbase. and cases where positive homotropic cooperativity and negative co-operativity occur. Fig 12.5 You should be able to accurately distinguish between cases where there is no interaction between binding sites. This regulation takes place in different ways.2 You should be able give a detailed discussion of the Monod-Wyman-Changeux (MWC) model.3 You should be able to describe the assumptions leading to the Hill equation and the Hill equation itself. You should also be able to distinguish between no co-operativity.4 You should be able to deduce and describe the Adair equation for the binding of a ligand to a protein with more than one binding site for that ligand. as well as the concept ``fractional saturation'' (Y). homotropic and heterotropic co-operativity by referring to the processes and their results (kinetics) (pp 224^225). You should know the uses and limitations of this equation (pp 228^229). you should be able to describe ways in which the relationship between Yand [S].
1 The regulation of ATCase and Pyruvate dehydrogenese is described on page 269 and 272 respectively.3. Explain the relationship between the latter three constants and Kb1 and Kb2 at no co-operativity.3.3. as well as other factors affecting metabolic regulation (pp 263^269).2.1 The course of the reaction.4 Briefly describe the relationship between allosteric enzymes and the rapid increase of metabolic regulation. positive co-operativity and negative co-operativity. 7 . as well as the factors controlling enzyme concentration in vivo (p 258). 8. In this case you do not have to study the kinetics discussed (pp 259^260).2 The explanation of co-operativity given by this model.3. 7 .3 The way in which allosteric regulation takes place according to this model.4 The KNF model and allosteric regulation. These examples are compulsory and may be used in assessment.1 The assumptions regarding the course of the reaction and the constants involved.2 Get an overview of the properties of the metabolic pathways that function in steady states. 8 THE SIGNIFICANCE OF SIGMOIDAL BEHAVIOUR 8. 7. Kb and the intrinsic constants KTT.2. .4 Sigmoidal kinetics can be observed in the absence of co-operativity. and the distinction between K-series and V-series enzymes.3 Briefly examine the regulation metabolics that operate under conditions of of equilibrium by controlling enzyme activity (pp 262^263).2 The equation for Y in this model.) 7 . 7. 7 .2. positive co-operativity and negative co-operativity. 9 SELF-EVALUATION 9. concentrating on the mechanism (pp 250^252). Three scenarios are mentioned. 8. KRT and KRR. referring to the following main points (pp 246^249): 7 . Discuss each scenario.1 Discuss the role of allosterics in the regulation van metabolism by referring to the environment in which enzymes occur. 7 .Take note of these two concrete examples of enzyme regulation to help you conceptualise the theoretical basis they are built on. 8.3 The constants Kt.3 Now compare the Koshland-Nemethy-Filmer (KNF) model. the relationship with the and the relationship between Kb1 and Kb2 at no co-operativity. (You do not have to know the deduction for Y in this case.20 7 .
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