# 2008 University of South Africa

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University of South Africa
Muckleneuk, Pretoria

BCH3711/1/2009^2011

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 (iii) BCH3711/1/2009^2011

Contents

Learning unit Page

Welcome! (v)

1 ENZYME NOMENCLATURE AND THE STRUCTURE AND FUNCTION OF

ENZYMES 1
1 Time 1
2 Textbook reference 1
3 Learning outcomes 1
4 Background to learning unit 1
5 Introduction to enzymology 2
6 The structure of protein 2
7 Specifities of enzyme functioning 2
8 Monomeric and oligomeric enzymes 3
9 Self-evaluation 3

2 BIOENERGETICS, CATALYSIS AND KINETICS 4

1 Time 4
2 Textbook reference 4
3 Learning outcomes 4
4 Background to learning unit 4
5 General concepts in bioenergetics 4
6 Factors affecting the rate of chemical reactions 5
7 The kinetics of uncatalysed and enzyme-catalysed reactions 5
8 The nature of enzyme-catalysed reactions 5
9 Self-evaluation 5

3 KINETICS OF SINGLE-SUBSTRATE ENZYME-CATALYSED REACTIONS 6

1 Time 6
2 Textbook reference 6
3 Learning outcomes 6
4 Background to learning unit 6
5 The relationship between vo and [So] 7
6 Rapid-reaction kinetics and relaxation kinetics 7
7 Self-evaluation and additional sources 7

4 ENZYME INHIBITION 9
1 Time 9
2 Textbook reference 9
3 Learning outcomes 9
4 Background to learning unit 9
5 Reversible inhibitors 10
6 Irreversible inhibitors 10
7 Self-evaluation and additional sources 11
 (iv)

Learning unit Page

5 KINETICS OF MULTISUBSTRATE ENZYME-CATALYSED REACTIONS 12

1 Time 12
2 Textbook reference 12
3 Learning outcomes 12
4 Background to learning unit 12
5 Reaction mechanisms of multisubstrate enzyme-catalysed reactions 13
6 Steady-state condition kinetics 13
7 Experimental approaches to the investigation of multisubstrate enzyme-
catalysed reactions 13
8 Self-evaluation and additional sources 13

6 THE ACTIVE CENTRE AND THE CHEMICAL NATURE OF ENZYME

CATALYSIS 15
1 Time 15
2 Textbook reference 15
3 Learning outcomes 15
4 Background to learning unit 15
5 Identifying the active site of enzymes 16
6 Mechanisms of catalysis 16
7 The use of co-factors in catalysis 16
8 Self-evaluation 16

7 ALLOSTERIC ENZYMES AND THE SIGNIFICANCE OF SIGMOIDAL

KINETICS 18
1 Time 18
2 Textbook reference 18
3 Learning outcomes 18
4 Background to learning unit 18
5 Binding of ligands to proteins 19
6 Mechanisms of catalysis 19
7 Sigmoidal kinetics and allosteric enzymes 19
8 The significance of sigmoidal behaviour 20
9 Self-evaluation 20
 (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.We are excited about introdu-
cing you to this subject, as it largely forms the basis of your grasp of biochemistry as
a whole.

This course also supports your other third year biochemistry modules ö metabo-
lism and molecular biology ö as enzymes are the catalysts in all biochemical pro-
cesses. The knowledge of basic biochemical concepts, especially protein
chemistry, that you gained in your second year modules, will stand you in good
stead here. The focus points of this course are general principles of enzyme func-
tioning, enzyme kinetics and the role of enzymes in metabolism. These topics have
great relevance for you as a student of natural sciences, as it will give you insight
into the nature and role of enzymes in living systems.Our purpose is also to demon-
state to you how the properties of enzymes kan be used for various applications in
natural science and biotechnology (BCH3714).

We trust that you will find this introduction to enzymology not only instructive and
interesting, but also highly enjoyable!

. Correspondence
Address all correspondence to:
The Registrar (Academic)
PO Box 392
UNISA
0003

. Tutorial letters and study guides

If you have received no tutorial letters or study guides, please contact the Depart-
ment of Despatch at the main Unisa campus in Pretoria. For telephonic enquiries,
use only the telephone number given on the inventory letter. Written enquiries
should be clearly marked for the attention of the Department of Despatch. No tutor-
ial matter can be obtained from the Department of Life Sciences in Potchefstroom.

Correspondence addressed to the same department may be placed in one envel-

ope. Please note, however, that correspondence addressed to different depart-
ments should be placed in different envelopes, each marked clearly for the
attention of the department involved. It is very important to write your student num-
ber, subject and course code clearly in the top right hand corner of all correspon-
dence.

. Administrative enquiries
All administrative enquiries, such as enquiries about changes of address and phone
numbers, should be sent directly to the Registrar, Unisa. All telephonic administra-
 (vi)

tive enquiries should be made to the relevant sections. Consult the booklet Unisa:
services and procedures, which you received with your study material at registra-
tion.

STUDY MATERIAL
The textbook for this module is:
(The contents of the first and second edition are exactly the same, but the second
edition may be the only one available. Please note that the information in the study
guide is based on the page numbers of the first edition.)
Bonner, P & Palmer, T. 2007. Enzymes: Biochemistry, Biotechnology, Clinical Chem-
istry. 2nd edition. Chichester, West Sussex, England: Hor wood, ISBN
1904275273.
OR
Palmer, T. 2001. Enzymes: Biochemistry, Biotechnology, Clinical Chemistry. 1st edi-
tion. Chichester,West Sussex, England: Horwood, ISBN 1898563780.

Additional sources (books) as well as a variety of internet sources are recom-

mended in the different learning units.We would like to encourage you to gain inter-
net access for this module, as it can be a good source of information. This includes
databases, literature search facilities and interactive websites.

EVALUATION

Examination
One two hour paper will be written in November. The date, time and examination
centre are indicated in Part 1, Section C of the Unisa Calender, and you will receive
written confirmation in September. You have to obtain 50% to pass the examination.

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. Tutorial Letter 101 for BCH3111 con-
tains the assignments and information about the closing dates and credits.

PRACTICALS AND PRACTICAL EXAMINATIONS

BCH3711 is a theory module, and no practical work will be done in this module.
However, certain aspects of this module forms part of the practical module
BCH3714. Please note that practical work and theory in Biochemistry are an entity,
and as such contribute to the attainment of outcomes. During the practicals theory
becomes reality when specimens, processes, concepts, ideas, systems, structures,
et cetera, are concretely handled. It is very important, therefore, that you work
through the program systematically and ö in the the form of reports, results, draw-
ings, graphs, et cetera ö keep accurate records of the practical work you have
 (vii) BCH3711/1

done. Make it your goal to ensure that you attain all the outcomes formulated at the
start.

A sound theoretical background is essential for success in BCH3714 , which will be

presented at the Potchefstroom campus of the Northwest University in September.

ACTION WORDS
Questions, whether in assignments or examinations, always contain certain key or
action words.You should know what these action words mean and what is required
when you answer the questions. To assist you in this, a short list of these words is
supplied below.

. Name
Just write down the bare facts as briefly as possible.

. Describe
Here we require you to demonstrate your knowledge and to relate properties, facts
or results in a logical, well-structured way. No commentary or discussion is neces-
sary.

. Define
Reproduction of knowledge is required, which involves a clear, concise and author-
itative description of a concept to demonstrate its meaning in no uncertain terms.

. Provide an overview
An extensive volume of knowledge should be summarised logically and systemati-
cally and conveyed without losing sight of the essence of the matter.

. Explain
Formulate the concept as simply as possible to ensure that the reader understands
it. Make use of illustrations, descriptions and examples, and give reasons for state-
ments and results.

. Prove
Substantiate the facts by logically presenting acceptable facts.

. Compare
You have to be careful here. Do not describe or discuss one matter fully before you
move on to the next. Facts, incidents or problems should be contrasted throughout,
indicating similarities and differences.
 (viii)

. Discuss
Discussion presupposes insight and discernment when it comes to application and
judgement. Here we expect you to analytically investigate and discuss different as-
pects of the matter or statement.

. Analyse
The contents are divided into parts or elements and discussed. Causes and results
are detected and relationships determined.

. Evaluate
Here we expect you to judge something on the basis of certain criteria and express
a well-motivated value judgement.

GENERAL OUTCOMES
The South African Qualifications Authority Act 58 of 1995 requires university stu-
dents to achieve the following outcomes at third year level:
. Competence. 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, re-
gulations, principles and theories, and be able to place your knowledge and
understanding within the broader framework of biological and other know-
ledge
ö know the basic applications, 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 ana-
lyses and syntheses of quantitative and/or qualitative data

. Autonomous learning. This requires you to:

ö function in a fluctuating and unfamiliar learning environment by using your
initiative and being responsible
ö evaluate yourself, and your ability to address your learning needs, astutely
and correctly

MODULE PLAN
This module is divided into the following seven learning units:
Learning unit 1 Enzyme nomenclature and the structure and function of enzymes
Learning unit 2 Bioenergetics, catalysis and kinetics
Learning unit 3 Kinetics of single-substrate enzyme-catalysed reactions
Learning unit 4 Enzyme inhibition
Learning unit 5 Kinetics of multisubstrate enzyme-catalysed reactions
Learning unit 6 The active centre and chemical nature of enzyme catalysis
Learning unit 7 Allosteric enzymes and the meaning of sigmoidal kinetics
 (ix) BCH3711/1

MODULE OUTCOMES
By the end of this module, you will be conversant with
. the nomenclature of enzymes
. the concepts of catalysis and kinetics of single- and multi-substrate enzyme-
catalysed reactions
. enzyme inhibition and the different mechanisms
. the determination and meaning of enzyme-kinetic parameters
. experimental approaches to enzyme kinetics, data processing and data
interpretation
. the properties of allosteric enzymes, the sigmoidal behaviour of enzymes and
their importance with respect to metabolic regulation
 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.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistr y,

Biotechnology, Clinical Chemistry (2nd edition), Chapters 1±5.

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
. demonstrate a well-rounded and systematic knowledge of the structural
aspects of enzymes and protein
. 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,
although enzyme preparations have been used by humans in processes such as
fermentation for millenia. Apart from their role as catalysts in all biological pro-
cesses, enzymes have become indispensable to the biomedical profession and in-
 2

dustry.The first enzymes were described early in the 20th century, 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, and the use of recombinant en-
zymes has become commonplace in medicine and industry. In this learning unit
we will give an overview of the history of the study of enzymes and general termi-
nology.

5 INTRODUCTION TO ENZYMOLOGY
5.1 Study the descriptions of enzymes in chapter 1, page 3, as well as the mean-
ings of the terms ``substrate'', ``product'', ``co-factor'', ``apoenzyme'', ``holoenzyme'',
``coenzyme'' and ``prosthetic group'' to enable you to give a description of each
and draw comparisons.
5.2 You should be able to provide an overview of the history of the study of
enzymes (pp 3^4).
5.3 The classification of enzymes according to the system of the IUBMB's Enzyme
Commission is of importance here. You should be able to distinguish clearly
between the six classes of enzymes according to naming, numbering and type
of reaction they catalyse ö we expect you to be thorough and give evidence of
insight into the system.You should be able to classify an enzyme reaction in the
group to which it belongs, and should understand the numbering system of the
IUBMB's Enzyme Commission. The examples given in the textbook should be
studied (pp 4^12).

6 THE STRUCTURE OF PROTEIN

6.1 A basic knowledge of protein structure is vital in enzymology. It is important that
you know what is meant by conjugated proteins and that you are able to give
examples of these (p 15).
6.2 In order to understand the functioning of enzymes, you should have a general
knowledge of the structure and properties of amino acids and the levels of
protein structure. This was fully dealt with in your second year, so we expect
you to have a sound and systematic knowledge of this topic. Use the textbook
to refresh your memory (pp 16^56).
6.3 You can study an outline of the acid-based properties of protein (pp 56^57).
6.4 You should know the meaning of the terms ``salting in'', ``salting out'', ``ionic
strength'', ``insoluble complexes'', ``denaturation'', and ``isoelectric point'', and the
effect of salts, pH and temperature.You should also know the biochemical prin-
ciples underlying these processes. Please pay particular attention to Figure
3.12 and Figure 3.11 (pp 61^64).

7 SPECIFITIES OF ENZYME FUNCTIONING

7.1 You should be able to distinguish between the type specifities of enzymes
(p 67).
7.2 You should be able to describe the components and general properties of the
active centre. You should have insight into the impact of changes in external
 3 BCH3711/1

factors such as pH, temperature and ion strength upon these components
(pp 68^70).
7.3 With reference to the components of the active centre studied in 7.2, you
should be able to describe the different hypotheses regarding enzyme func-
tioning and discuss the respective aspects that are addressed by these
hypotheses and those that are not addressed (Fig 4.2^4, pp 70^73).

8 MONOMERIC AND OLIGOMERIC ENZYMES

8.1 You should be able to distinguish between monomeric and oligomeric
enzymes with respect to structure and by means of relevant examples
(pp 76^84).

9 SELF-EVALUATION
9.1 Problems 1.1 and 1.2 on pages 12 to 14 are focussed on the classification of
enzymes. 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.
After solving these problems, find the answers at the back of your text-
book and compare them with your answers.

9.2 To test your general knowledge of protein structure after studying it, write a
summary of about 20 lines about the structure of protein. Now compare it with
the summary at the end of chapter 3 (p 64). Do the same exercise after study-
ing chapter 4. This will help you to evaluate your knowledge of and insight into
the third and fourth learning outcomes.
9.3 Visit the website of the Enzyme Commission of the International Union of
Biochemistry and Molecular Biology (IUBMB) at http://www.chem.qmw.ac.uk/
iubmb/kinetics/ and compare the terminology and classification of enzymes
compiled by this commission in 1981 with those in your textbook. This is not
compulsory, 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).
 4

Learning unit

2 BIOENERGETICS, CATALYSIS AND

KINETICS

1 TIME

You need about 16 hours to complete this learning unit successfully.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistr y,

Biotechnology, Clinical Chemistry. 2nd edition, Chapter 6.

3 LEARNING OUTCOMES

By the end of this learning unit you should be able to

. 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 en-
zyme kinetics. This includes the laws of thermodynamics, energy and reaction rate.
It also forms the basis of the next three learning units, where enzyme kinetics will be
investigated, and outlines the background and principles underlying enzyme ki-
netics. It also serves as critically important background knowledge for the next
learning units.

5 GENERAL CONCEPTS IN BIOENERGETICS

5.1 You should gain an understanding of and insight into the following concepts:
 5 BCH3711/1

the First and Second Laws of Thermodynamics, enthalpy (DH), enthropy (DS),
free energy (DG) and standard free energy of change (DGK) (pp 87^90).
5.2 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
is suited to the role it fulfils (pp 90^91).

6 FACTORS AFFECTING THE RATE OF CHEMICAL REACTIONS

6.1 You should know the three factors that affect the rate of chemical reactions,
and be able to describe them. Make sure that you can distinguish between
the terms ``collision theory'', ``activation energy;'' (Fig 6.1) and ``transitional state''
(Fig 6.1^2, pp 91^95).

7 THE KINETICS OF UNCATALYSED AND ENZYME-CATALYSED REAC-

TIONS
7.1 You should be able to discuss the kinetics of uncatalysed reactions, and should
understand the Principle of Mass Action and the concept of reaction order,
which is based on it. 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).
7.2 Get an overview of the historical introduction to the kinetics of enzyme-cata-
lysed reactions (p 98).
7.3 You should be able to describe the relation between vo and [So ] at constants
[Eo ] and the order in which the reaction takes place, and to give reaction equa-
tions for vo and Vmax (pp 99^100).

8 THE NATURE OF ENZYME-CATALYSED REACTIONS

8.1 Take note of the methods used to investigate the kinetics of enzyme-catalysed
reactions. Pay special attention to initial reaction studies, as they will be used
later in this module (pp 100^104).
8.2 You should be able to describe and explain the course followed by the free-
energy profile of an enzyme-catalysed reaction in which a substrate and
enzyme product complex is formed.

9 SELF-EVALUATION
9.1 For self-evaluation, do problems 6.1 and 6.2 on page 105 in the textbook. The
answers are given at the back of your textbook.
 6

Learning unit

3 KINETICS OF SINGLE-SUBSTRATE
ENZYME-CATALYSED REACTIONS

1 TIME

You need about 16 hours to complete this learning unit successfully.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistr y,

Biotechnology, Clinical Chemistry. 2nd edition, Chapter 7.

3 LEARNING OUTCOMES

By the end of this learning unit you should be able to

. describe the relationship between initial reaction rate and substrate
concentration as defined by Henri, Michaelis and Menten, and identify the
assumptions on which this relationship is based
. describe the principles underlying steady-state kinetics as presented by
Briggs and Haldane, and derive the Michaelis-Menten equation
. determine and explain the meaning of the most common enzyme-kinetic
parameters by means of problems
. 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 sub-
strate specificity. However, it is also necessary to describe how rapidly and effec-
tively an enzyme can work to bind its substrate and turn it into product. Certain
kinetic paramaters are used here, and in this learning unit we investigate the rela-
tively simple kinetics of single-substrate reactions. We also examine the assump-
 7 BCH3711/1

tions we have to make in order to describe the vitally important relationship be-
tween initial reaction rate vo and substrate concentration [So ]. These assumptions
are important, as they are of special value when it comes to experimental investiga-
tion.You will also be introduced to the way in which the most common kinetic para-
meters such as vo, Vmax, Km and Kcat are calculated, and you will learn what these
parameters mean.

5 THE RELATIONSHIP BETWEEN vo AND [SO]

5.1 Steady-state kinetics is based on the six commonly used assumptions as
defined by Henri, Michaelis and Menten. You should be able to identify and
describe these assumptions, which were derived from an equilibrium state
view of a single substrate reaction. You should also be able to fully describe
the deductions made from them, and indicate the relationship between vo and
[So ] (Michaelis-Menten equation) (pp 107^109).
5.2 You should be able to describe the adaptations of the Michaelis-Menton
deductions suggested by Briggs and Haldane, namely to adopt a steady-state
approach to the enzyme-substrate complex.You should also be able to identify
the differences between the two approaches, and to describe and apply the
adaptation of the equation for vo (pp 109^111).
5.3 You should be able to fully describe the meaning, units and calculation of the
parameters Vmax, Ks, Km, Kcat and Kcat/Km, and to solve problems in which these
parameters are used (pp 111^113).
5.4 The use of primary graphic methods to determine the values of Km and Vmax is
very important. We confine ourselves to three approaches: those of Linewea-
ver-Burk, Eadie-Hofstee, and Hanes. In each of these cases you should know
which modification of the Michaelis-Menten equation was carried out, and be
able to apply the graphic representation in order to calculate Km and Vmax. Note
the possible advantages and disadvantages of each of the three methods, 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).

6 RAPID-REACTION KINETICS AND RELAXATION KINETICS

6.1 Get an overview of the use of rapid-reaction kinetics as well its concomitant
measuring techniques, and take note of the limitations of thermodynamics in
the study of metabolism (pp 118^110).
6.2 You should be able to present the theoretical course of single substrate reac-
tions and to state the meaning of the terms ``induction period'', ``pre^steady
state'' and ``steady state'' in their respective contexts (pp 120^121).
6.3 You should be able to give an overview or the meaning of relaxation kinetics
and its applications (pp 122^123).

7 SELF-EVALUATION AND ADDITIONAL SOURCES

7.1 The terms used in enzyme kinetics sometimes differs from those that are
commonly used. The terminology used in your textbook will suffice, but it's a
 8

good idea to acquaint yourself with the conventions regarding notation in

enzymology by visiting the website of the IUBMB at
http://www.chem.qmw.ac.uk/iubmb/kinetics/.
7.2 For self-evaluation, do problems 7.1 and 7.2 on page 127 in the textbook. The
answers are given at the back of the textbook, but please note that you should
do the calculation of vo in problem 7.2 graphically before you can proceed to
the next step when solving this problem. NB: always use graph paper!
7.3 If you have access to a library, you can consult the following sources to supple-
ment your reading or to do extra enzyme kinetics problems for self-evaluation
(not compulsory):
. Purich, DL (ed). 1983. Contemporary enzyme kinetics and mechanism. New
York: Academic Press, ISBN 0125680503.
. Engel, PC. 1981. Enzyme kinetics: the steady-state approach. 2nd edition.
London: Chapman & Hall, ISBN 0412239701.
. Christensen, HN & Palmer, GA.1974. Enzyme kinetics: a learning program for
students of the biological and medical sciences. 2nd edition. London: WB
Saunders, ISBN 0721625916.
 9 BCH3711/1

Learning unit

4 ENZYME INHIBITION

1 TIME

You need about 12 hours to complete this learning unit successfully.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistry,Biotechnology,

Clinical Chemistry. 2nd edition, Chapter 8.

3 LEARNING OUTCOMES

By the end of this learning unit you should be able to

. recognise the role of enzyme inhibitors in metabolism
. describe the classification and functioning of reversible and irreversible
enzyme inhibitors by citing examples
. to describe and execute the methods used when investigating the
identification and kinetics of enzyme inhibitors, 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. Enzymes were created in
such a way that they can be regulated by particular inhibitors. If biological reactions
are not regulated, several pathological conditions, such as scurvy, can develop (de-
ficiency in the regulation of lycil oxidase by Vit C). Enzyme inhibitors are also de-
signed and used in the pharmaceutical industry to suppress certain processes.
Antibiotics, which usually act as inhibitors of a bacterial replication, transcription or
translation process, are a classical example of this. On the other hand, inhibitors ö
especially irreversible inhibitors ö can stop certain reactions, which can lead to cell
 10

death. Several toxins work in this way. Therefore, it is very important to thoroughly
investigate this phenomenon; it is one of the aspects of enzymology that gives us
more insight into metabolic regulation, and that can be harnessed by medical
science and industry.

5 REVERSIBLE INHIBITORS
5.1 You should be able to accurately distinguish between the properties of reversi-
ble and irreversible inhibitors with reference to the examples in the textbook
(p 129).
5.2 You should also be able to accurately distinguish between the types of reversi-
ble inhibitors. Use the following to guide you (pp 128^149):
5.2.1 The inhibition mechanism, that is, where the inhibitor binds to the
enzyme, and the structure of the inhibitor are important (Fig 8.2). You
should be able to give an example of such an inhibitor, and to make a
deduction as to the possible mechanism of an inhibitor on the basis of
structural data regarding the inhibitor.
5.2.2 You should be able to describe the equilibrium-state kinetics of the
different inhibitors by referring to the deviation caused to the Michaelis-
Menten equation, and to describe or predict and explain the effect of the
inhibitor on the Km and Vmax values.
5.2.3 You should be able to describe the Lineweaver-Burk equation in the
presence of the inhibitor, and describe or predict the Lineweaver-Burke
graph and the effect of the inhibitor on its course. Using the kinetic data,
you should be able to identify the type of inhibitor by means of the Line-
weaver-Burk graph.
5.2.4 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), and to calculate the inhibition constants
Ki and KI by using the Michaelis-Menten equation.
5.2.5 You should have the cognitive skills to process experimental data
correctly, do correct calculations and make motivated deductions about
the nature and mechanism of enzyme inhibition. A problem-solving
approach will be followed in the evaluation of your abilities.

5.3 You should be able to compare partial, substrate and allosteric inhibition (all
reversible inhibition mechanisms) in outline form (pp 148^149).

6 IRREVERSIBLE INHIBITORS
6.1 You should be able to describe the nature of this type of inhibition by referring
to examples, and give a short summary of its effect on Km and Vmax values.You
should also be able to distinguish between reversible and irreversible inhibitors
on the basis of binding properties and kinetic parameters. In other words, you
should be able to explain when an inhibitor should be regarded as irreversible
(pp 149^151).
 11 BCH3711/1

7 SELF-EVALUATION AND ADDITIONAL SOURCES

7.1 Pay the website of the Enzyme Commission of the International Union of
Biochemistry and Molecular Biology (IUBMB) another visit at http://
www.chem.qmw.ac.uk/iubmb/kinetics/ and compare the classification and
kinetics of inhibitors with those in your textbook.This is not compulsory, but will
help you to obtain a proper overview of the classification of enzyme inhibitors.
You can also consult the additional sources listed in the previous learning unit.
7.2 For self-evaluation, do problems 8.1 to 8.4 on pages 152 to 153. The answers are
given at the back of the textbook. Please note that problem 8.3 alludes to
problem 7.2 in the previous learning unit. These questions test your ability to
achieve the third learning outcome of this learning unit.
7.3 Investigate and compare the mechanisms of the following inhibitors: DFP,
cyanide, and ethylene glycol.
 12

Learning unit

5 KINETICS OF MULTISUBSTRATE
ENZYME-CATALYSED REACTIONS

1 TIME

You need about 10 hours to complete this learning unit successfully.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistr y,

Biotechnology, Clinical Chemistry. 2nd edition, Chapter 9.

3 LEARNING OUTCOMES

By the end of this learning unit you should be able to

. classify, describe and distinguish the different multisubstrate enzyme-
catalysed reaction mechanisms
. describe and explain the use of steady state kinetics and product inhibitors
in the experimental investigation of multisubstrate enzyme-catalysed
reaction mechanisms, 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, which in ki-
netics is seen as an additional substrate. Therefore it is necessary to investigate
and describe this type of reaction. Also, the kinetics is more complex, and one
needs to examine the experimental approach to these mechanisms.
 13 BCH3711/1

5 REACTION MECHANISMS OF MULTISUBSTRATE ENZYME-CATA-

LYSED REACTIONS
5.1 Make sure that you can accurately distinguish between the different types of
sequential and nonsequential reaction mechanisms. Refer to the course of
the reaction and the formation of intermediary complexes in each case. You
should be able to identify a reaction mechanism on the basis of given data
(pp 155^156).

6 STEADY-STATE KINETICS
6.1 Steady-state kinetics can be applied in the case of multisubstrate reactions.
Here you should be able to describe Alberty's general rate equation and the
conditions under which this equation is valid (pp 157-158).
6.2 You should be able to describe the application of Alberty's general rate equa-
tion at high second substrate concentrations, [Bo ] of [AXo ] and Alberty's equa-
tion for a ping-pong bi-bi-mechanism respectively (pp 157-159).
6.3 You should be able to calculate kinetic parameters (Km- and Vmax values)
using Alberty's general rate equation, (ie steady-state kinetics) by means of
primary graphs from experimental data (p 159). This is comparable to the work
done in the case of Michaelis and Menten in learning unit 3.
6.4 You should be able to describe Dalziel's general rate equation and the purpose
it serves, and to use secondary graphs to calculate Km values in two-substrate
reactions (pp 159^160).

7 EXPERIMENTAL APPROACHES TO THE INVESTIGATION OF MULTI-

SUBSTRATE ENZYME-CATALYSED REACTIONS
7.1 The mechanism of a reaction is commonly determined by means of two
approaches: the use of primary graphs and the use of inhibitors. You should
be able to describe and explain the theoretical basis of the use of primary
graphs for this purpose, as well as the deductions made from these graphs. In
Figure 9.3 the differences seen here are clearly distinguished (pp 162^163).
7.2 You should also be able to indicate the possible uses of inhibitors by referring
to Cleland's two rules and their significance. You should also be able to point
out the relation between a variable substrate, an inhibitor, the reaction mechan-
ism and the kinetic course a reaction follows. Here you should make use of the
enumerative table on page 66, and be able to make deductions from experi-
mental data in this regard. Dit is extremely important that you understand this
table (p 166) well and are able to explain it (pp 163-167).

8 SELF-EVALUATION AND ADDITIONAL SOURCES

8.1 To test your knowledge of multisubstrate enzyme-catalysed reaction mechan-
isms after studying them, draw a diagram in which you classify the types of
mechanisms and reactions. Make use of the notation employed in the textbook
(substrates AX and B with products A and BX in a transferase-type reaction) for
 14

each of the mechanisms. Now compare your classification with the one in the
textbook. This will help you to get used to the way in which notation of the reac-
tions and classification of the mechanisms are done (first learning outcome).
8.2 Also see the classification done by the IUBMB at http://www.chem.qmw.ac.uk/
iubmb/kinetics/ if you have access to tye internet, or consult the additional
sources listed in learning unit 3.
8.3 For self-evaluation and to determine whether you have achieved the second
learning outcome, do problems 9.1 and 9.2 on pages 170 to 171. It is of the
utmost importance that you should be able to solve these problems. The
answers are given at the back of the textbook. Please note that these problems
make use of a combination of the two experimental approaches described in
Section 7.1. Carefully note the graphic representations of your choice of vari-
able substrates. Have a good look at the tabled data you received before
making your selection.
 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.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistr y,

Biotechnology, Clinical Chemistry. 2nd edition, Chapters 10 and 11.

3 LEARNING OUTCOMES

By the end of this learning unit you should be able to

. give a concise description of the different empirical methods used to
investigate the active centre
. give an onverview of the different catalytic mechanisms involved in
enzymes, and describe two examples of catalytic mechanisms in
enzymes
. 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. Certain amino
acids in the catalytic sites, as well as co-factors that may be involved, mediate these
interactions. In this learning unit we study the ways in which these interactions are
investigated.We also examine the chemical nature of enzyme catalysis and the role
 16

of co-factors with reference to examples.This knowledge will give us a better idea of

the catalytic functioning of enzymes.

5 IDENTIFYING THE ACTIVE SITE OF ENZYMES

5.1 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. In
each case, make use of an example to illlustrate the technique (pp 175^187).

6 MECHANISMS OF CATALYSIS
6.1 You should be able to distinguish between the chemical processes of acid-
based, electrostatic, covalent and enzyme catalysis, and to apply this informa-
tion to describe the catalytic mechanism of the enzymes chymotrypsin and
lysozyme (pp 193^200).

7 THE USE OF CO-FACTORS IN CATALYSIS

7.1 You should be able to compare the properties and role of metal-activated
enzymes with those of metallo-enzymes (pp 202^203).
7.2 Co-enzymes are used by several enzymes.You should be able to compare the
following co-enzymes with regard to origins, structure, and the catalytic role
they fulfil in enzyme reactions. You should also be able to give one example of
a situation in which enzyme reactions employ co-enzymes (pp 206^221):
7.2.1 Nicotinamide nucleotides
7.2.2 Flavine nucleotides
7.2.3 Adenosine phosphates
7.2.4 Co-enzyme A
7.2.5 Biotin
7.2.6 Co-enzyme B12

8 SELF-EVALUATION
8.1 This assignment will really help you to gain insight into the functioning of
enzymes by means of concrete examples, and to achieve the outcomes of this
learning unit. Select any two (or more, 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 (8.1.1^8.1.6) on these enzymes. You can choose any
organism, but if you have doubts, use the information on these enzymes in
domesticated bovine animals (Bos Taurus) or rats (Rattus Norvegicus). Possi-
ble internet sources are also listed below, although you can use other sources
as well.
. Chymotrypsyn (EC 3.4.21.1)
. Pyruvate dehydrogenase (EC 1.2.4.1)
. Lysozyme (EC 3.2.1.17)
. Hexokinase (EC 2.7.1.1)
. Glutathione reductase (EC 1.8.1.7)
 17 BCH3711/1

8.1.1 Systemic and common name

8.1.2 Reaction catalysed and type
8.1.3 Co-factors involved
8.1.4 Catalytic mechanism and type, as well as active site of enzyme
8.1.5 Metals, ions and inhibitors of enzyme
8.1.6 Functional parameters (ie kinetic parameters, optimal pH and tempera-
ture, pI)

Apart from your textbook (which contains limited information), good sources include
the following:
. BRENDA (http://www.brenda-enzymes.info/index.php4)
. EXPASY (http://au.expasy.org/enzyme/)
. KEGG (http://www.genome.ad.jp/kegg/)
. PUBMED (http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed)
. Methods in Enzymology. Series by Academic Press, ISBN 0121821862
 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.

2 TEXTBOOK REFERENCE

This learning unit is based on the textbook Enzymes: Biochemistr y,

Biotechnology, Clinical Chemistry. 2nd edition. Chapters 12, 13, and 14.

3 LEARNING OUTCOMES

By the end of this learning unit you should be able to

. describe the principles of ligand binding to a protein with regard to binding
constants and fractional saturation
. describe sigmoidal kinetics and co-operativity (different types) in
enzymes, as well as the molecular interactions they involve
. reproduce models describing sigmoidal kinetics and point out the
limitations of these models
. demonstrate insight into the significance of sigmoidal kinetics with regard
to enzymes in metabolic processes
. 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, flow of genetic information,
etc) is essential for the survival and functioning of the cell. Enzymes fulfil an active
role in this and have been constructed in such an extraordinary way that the regula-
 19 BCH3711/1

tion of activities adapts itself to the environment and needs of the cell and organism.
This regulation takes place in different ways, and in this learning unit you will be-
come better acquainted with these ways.

5 BINDING OF LIGANDS TO PROTEINS

5.1 You should be able to define the binding constant Kb for a protein with a single-
ligand binding site, as well as the concept ``fractional saturation'' (Y).You should
also be able to describe the equation for Y in steady states (pp 223^224).
5.2 You should be able to distinguish between positive, negative, homotropic and
heterotropic co-operativity by referring to the processes and their results
(kinetics) (pp 224^225).
5.3 You should be able to describe the assumptions leading to the Hill equation
and the Hill equation itself.You should also be able to explain how one uses this
equation in the Hill graph and indicate how deductions can be made from it
(pp 225^228).
5.4 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. You
should know the uses and limitations of this equation (pp 228^229).
5.5 You should be able to accurately distinguish between cases where there is no
interaction between binding sites, and cases where positive homotropic co-
operativity and negative co-operativity occur. In each case, refer to the rela-
tionship between the binding constants Kb1 and Kb2 in your equation. You
should also be able to distinguish between no co-operativity, positive co-oper-
ativity and negative cooperativity, by means of a graph and making use of the
relationship between Yand [S] (pp 229^232, Fig 12.4 a^d).
5.6 In order to illustrate the way in which co-operativity is typically investigated, you
should be able to describe ways in which the relationship between Yand [S], vo
and [So ], and the Scatchard plots respectively can be used. Please make a
special note of the conditions in which each of these three appraoches can
be used (pp 233^236).

6 MECHANISMS OF CATALYSIS
6.1 You should be able to distinguish between the biochemical processes of acid-
base, electrostatic, covalent and enzyme catalysis, and to apply this informa-
tion in describing the catalytic mechanism of the enzymes chymotrypsyn and
lysozyme (pp 193^200).

7 SIGMOIDAL KINETICS AND ALLOSTERIC ENZYMES

7.1 You should be able to define the term ``allosterism'' and describe the role of
allosterism in the regulation of enzyme activities. Distinguish between the so-
called Tand R forms of a protein (pp 240^241).
7.2 You should be able give a detailed discussion of the Monod-Wyman-Changeux
(MWC) model, referring to the following main points (pp 241^246):
 20

7.2.1 The assumptions regarding the course of the reaction and the constants
involved. (You do not have to know the deduction for Y in this case.)
7.2.2 The explanation of co-operativity given by this model.
7.2.3 The way in which allosteric regulation takes place according to this
model, and the distinction between K-series and V-series enzymes.

7.3 Now compare the Koshland-Nemethy-Filmer (KNF) model, referring to the

following main points (pp 246^249):
7.3.1 The course of the reaction.
7.3.2 The equation for Y in this model, the relationship with the and the rela-
tionship between Kb1 and Kb2 at no co-operativity, positive co-operativ-
ity and negative co-operativity.
7.3.3 The constants Kt, Kb and the intrinsic constants KTT, KRT and KRR.
Explain the relationship between the latter three constants and Kb1 and
Kb2 at no co-operativity, positive co-operativity and negative co-opera-
tivity.
7.3.4 The KNF model and allosteric regulation.

7.4 Sigmoidal kinetics can be observed in the absence of co-operativity. Three

scenarios are mentioned. Discuss each scenario, concentrating on the
mechanism (pp 250^252).

8 THE SIGNIFICANCE OF SIGMOIDAL BEHAVIOUR

8.1 Discuss the role of allosterics in the regulation van metabolism by referring to
the environment in which enzymes occur, as well as the factors controlling
enzyme concentration in vivo (p 258).
8.2 Get an overview of the properties of the metabolic pathways that function in
steady states. In this case you do not have to study the kinetics discussed
(pp 259^260).
8.3 Briefly examine the regulation metabolics that operate under conditions of of
equilibrium by controlling enzyme activity (pp 262^263).
8.4 Briefly describe the relationship between allosteric enzymes and the rapid
increase of metabolic regulation, as well as other factors affecting metabolic
regulation (pp 263^269).

9 SELF-EVALUATION
9.1 The regulation of ATCase and Pyruvate dehydrogenese is described on page
269 and 272 respectively.Take note of these two concrete examples of enzyme
regulation to help you conceptualise the theoretical basis they are built on.
These examples are compulsory and may be used in assessment.