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From Enzyme Models to Model Enzymes 1st Edition.
Edition Anthony J. Kirby Digital Instant Download
Author(s): Anthony J. Kirby, Florian Hollfelder
ISBN(s): 9780854041756, 0854041753
Edition: 1st Edition.
File Details: PDF, 4.44 MB
Year: 2009
Language: english
From Enzyme Models to Model Enzymes
From Enzyme Models to Model
Enzymes

Anthony J. Kirby
University Chemical Laboratory, University of Cambridge, Cambridge, UK

Florian Hollfelder
Department of Biochemistry, University of Cambridge, Cambridge, UK
Cover picture. Graphic representation (by Dr. Jörg Harms) of the X-ray
structure of a bacterial 50S ribosome (Cell, 2001, 107, 679–688). This is the
larger subunit of the complex ‘‘machine’’ that translates the genetic code into
the specific primary sequence of amino-acids defining each natural protein
(Figure 1.1) – enzymes included. Significantly, the ribosome combines a few
RNA chains (silver ribbons) with many protein molecules (coloured), and can
be thought of as the prototype model enzyme. We thank Professor Ada Yonath
for supplying the graphics file.

ISBN: 978-0-85404-175-6

A catalogue record for this book is available from the British Library

r Anthony J. Kirby and Florian Hollfelder 2009

All rights reserved

Apart from fair dealing for the purposes of research for non-commercial purposes or for
private study, criticism or review, as permitted under the Copyright, Designs and Patents
Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not
be reproduced, stored or transmitted, in any form or by any means, without the prior
permission in writing of The Royal Society of Chemistry or the copyright owner, or in the
case of reproduction in accordance with the terms of licences issued by the Copyright
Licensing Agency in the UK, or in accordance with the terms of the licences issued by the
appropriate Reproduction Rights Organization outside the UK. Enquiries concerning
reproduction outside the terms stated here should be sent to The Royal Society of
Chemistry at the address printed on this page.

Published by The Royal Society of Chemistry,

Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 0WF, UK

Registered Charity Number 207890

For further information see our web site at www.rsc.org

Preface

We have been fascinated by enzymes since we were students in the 1960s and
the 1990s. In the early days the mantra – ‘‘there’s nothing magic about
enzymes’’ – simply added to the fascination. Of course ‘‘like magic’’ in these
postromantic times simply means (to quote the Oxford English Dictionary)
‘‘without any apparent explanation; with incredible rapidity’’: simple maybe,
but it’s hard to think of a more intriguing starting point for a research
career. And we still would like to understand – as well as possible – how
enzymes work.
At one level we can already explain enzyme catalysis: what an enzyme does is
bind, and so stabilize selectively, the transition state for its specific reaction. But
our current level of understanding fails the more severe, practical test – the
creation of new systems with catalytic efficiencies to rival those of natural
enzymes. As researchers, mostly in chemistry but at defining points in bio-
chemistry departments, our approach has been experimental: applying the
methods of contemporary organic chemistry, enzymology and more recently
molecular biology, to develop systems that model or – better – mimic enzymes:
and in particular their catalytic efficiency.
We settled on the title of this book only after some discussion, and some
definitions are in order. An enzyme model simply models an aspect, sometimes
more than one aspect, of the chemistry of a particular enzyme. At the next level,
we expect enzyme mimics to catalyze reactions of substrates free in solution, by
mechanisms that are demonstrably enzyme-like, involving both binding and
catalysis. However, to call the most successful systems artificial enzymes seems
no longer appropriate. As protein chemistry develops in countless new direc-
tions, existing protein structures can provide appropriate starting points for
new catalysts, which are not exactly artificial. We propose the term model
enzyme. Everyone expects that their model aeroplane should at least fly! And

From Enzyme Models to Model Enzymes

By Anthony J. Kirby and Florian Hollfelder
r Anthony J. Kirby and Florian Hollfelder 2009
Published by the Royal Society of Chemistry, www.rsc.org

v
vi Preface
ribozymes (Chapter 5), which qualify as successful systems, are serious candi-
dates to have been Nature’s models for all enzymes.
Our first intention was to write a short student text, introducing the area of
enzyme models for readers from the many disciplines that offer a basic
grounding in chemistry and biochemistry. The project evolved as we wrote,
reflecting the ways the subject is developing, to take advantage of the avail-
ability of new and powerful biological methodologies. The resulting text is still
short, and still intended to be accessible to the same readership: but attempts in
a final chapter to put recent developments in the perspective of the major body
of work that has gone before: to trace the underlying logic of these develop-
ments, and to identify the lessons to be learned. So that as we understand and
come to terms with the limitations of classical approaches to ‘‘design-based’’
model systems, we learn how to complement – and perhaps even replace – them
with new and emerging methods: based on the successful development of
enzymes by natural evolution, and using iterative approaches to make the best
use of design and selection. It is in the nature of science that the very recent
work we review may well be superseded by even more remarkable results in this
fast moving field.
We have been helped, encouraged and inspired by our students, colleagues
and teachers, too numerous to mention, from all over the world: but we must
acknowledge our special debts of gratitude to the late Bill Jencks, who could
turn physical organic chemistry into biological magic.

Tony Kirby, Florian Hollfelder

Cambridge, UK
Contents

Chapter 1 From Models Through Mimics to Artificial Enzymes 1

1.1 Introduction to Enzyme Chemistry 2

1.1.1 Why are Enzymes so Big? 3
1.1.2 Functional Groups Available to Enzymes 4
1.2 Principles of Catalysis by Enzymes 6
1.2.1 Dependence on pH 7
1.3 General Acid–Base Catalysis 8
1.3.1 Experimental Evidence 8
1.3.2 Mechanisms 9
1.3.3 Kinetic Equivalence 13
1.4 Intramolecularity 14
1.4.1 Efficiency of Intramolecular Catalysis 17
1.5 Energetics 18
1.6 Binding and Recognition 20
1.6.1 Hydrophobic Binding 22
1.6.2 The Special Environment of the Active Site 24
1.7 Cofactors 24
1.8 Many Enzymes Use the Same Basic Mechanisms 25
1.9 Enzyme Models, Mimics and Pretenders 26

Chapter 2 Evaluation of Catalytic Efficiency in Enzymes and

Enzyme Models 29

2.1 Introduction 29
2.2 Measurement of Uncatalyzed Rate Constants, kuncat 30
2.3 Characterizing the Catalytic Reactivity of an Enzyme
or an Enzyme Model 32
2.3.1 Comparing Catalyzed and Uncatalyzed Rates 34

From Enzyme Models to Model Enzymes

By Anthony J. Kirby and Florian Hollfelder
r Anthony J. Kirby and Florian Hollfelder 2009
Published by the Royal Society of Chemistry, www.rsc.org

vii
viii Contents

2.3.2 Calculating Rate Accelerations 35

2.4 Catalytic Efficiencies of Representative Enzymes:
The Size of the Challenge 41

Chapter 3 Constructing Enzyme Models – Building up Complexity 42

3.1 Solvents – Catalysis Without Functional Groups? 43

3.2 Introducing Catalytic Groups – Without
Positioning Them 46
3.3 Positioning of Substrate and Catalytic Groups
by Covalent Design 47
3.4 Binding the Ground State by Noncovalent
Interactions 47
3.5 Binding the TS More Strongly than the GS by
Noncovalent Interactions 50
3.6 Existing Enzymes as Catalytic Scaffolds
to Accommodate New Functions 52
3.6.1 Enzymes Modified by Addition of
Functionality 54
3.6.1.1 Exploiting Modular Build-Up
of Binding and Catalytic Features:
Chimeras of Binding Proteins and
Reactive Chemical Functionality 54
3.6.1.2 Noncovalent Introduction of
Reactive Cofactors: Transition-Metal
Catalysts in a Protein 55
3.6.1.3 Covalent Derivatization of
Active-Site Residues
to Introduce Reactive Cofactors 56
3.6.2 Site-Directed Mutants of Enzymes: Minimalist
Protein Redesign 56
3.6.3 Exploring Enzyme Promiscuity 57

Chapter 4 Enzyme Models Classified by Reaction 61

4.1 Acyl Transfer 61

4.1.1 The Serine Proteases. Typical Active Sites 62
4.1.1.1 The Active-Site Environment . . .
and Mechanism 64
4.1.1.2 Intramolecular Models 66
4.1.1.3 Supramolecular Models 69
4.1.1.3.1 Cyclodextrins 69
4.1.1.3.2 Synthetic Models 72
4.1.2 SH Hydrolases 75
4.1.2.1 Models 76
Contents ix

4.1.2.2 Intramolecular Models 78

4.1.2.3 Supramolecular Models 79
4.1.3 Aspartic Proteinases 79
4.1.3.1 Intramolecular Models 80
4.1.4 Metallopeptidases/Amide Hydrolases 83
4.1.4.1 Enzymes 86
4.1.4.2 Model Systems with One Metal
Centre 86
4.1.4.2.1 Intramolecular Reactions 88
4.1.4.2.2 Supramolecular
Metalloprotease/Peptidase Models 88
4.1.4.3 Model Systems with Two Metal
Centres 90
4.1.4.3.1 Aminopeptidase and
Lactamase Models 92
4.2 Phosphoryl Transfer 95
4.2.1 Phosphoryl Group Transfer from Monoesters 96
4.2.1.1 Enzymes I. Phosphoryl Transfer
Without Metals: PTPases 98
4.2.1.1.1 Models 100
4.2.1.1.2 Intramolecular Models 100
4.2.1.1.3 Supramolecular Models 101
4.2.1.2 Enzymes II. Metalloenzymes 102
4.2.1.2.1 Models 102
4.2.2 Phosphoryl Group Transfer from
Phosphodiesters 105
4.2.2.1 Intramolecular Reactions 106
4.2.2.1.1 Intramolecular Attack
by OH 108
4.2.2.2 Enzymes I. Phosphoryl Group
Transfer Without Metals 111
4.2.2.2.1 Transfer to
Neighbouring OH 113
4.2.2.2.2 Supramolecular Models 114
4.2.2.3 Enzymes II. Metalloenzymes 117
4.2.2.3.1 Supramolecular Models:
RNA Cleavage 118
4.2.2.3.2 Supramolecular Models:
DNA-Cleavage 122
4.3 Glycosyl Transfer 126
4.3.1 Simple Models 129
4.3.2 Enzymes 132
4.3.2.1 Glycoside Hydrolases 133
4.3.2.2 Glycoside Transferases 135
4.3.3 Intramolecular Models 138
4.3.4 Enzyme Mimics 142
x Contents

4.4 Hydrogen Transfer 145

Introduction 145
4.4.1 Enolization: Proton Transfer from Carbon 146
4.4.1.1 Simple Models 148
4.4.1.2 Intramolecular Models 149
4.4.1.3 Catalysis by Metal Ions 151
4.4.1.4 Enzymes Catalyzing Enolization 152
4.4.1.4.1 Triose Phosphate Isomerase 152
4.4.1.4.2 Citrate Synthase 155
4.4.1.4.3 The Enolase Superfamily 155
4.4.1.4.4 Mandelate Racemase 157
4.4.1.4.5 Models 157
4.4.2 Hydride Transfer 159
4.4.2.1 Uridine Diphosphate-galactose-4-
epimerase 160
4.4.2.2 Dehydrogenases 161
4.4.2.3 Models 161
4.4.2.4 Intramolecular Models 165
4.4.3 Hydrogen-Atom Transfer 165
4.5 Radical Reactions 168
4.5.1 Coenzyme Initiators Based on
Adenosylcobalamin 168
4.5.2 Radicals in Enzyme-Active Sites 171
4.5.2.1 Pyruvate-formate Lyase 171
4.5.2.2 Ribonucleotide Reductases 172
4.5.3 Models 174
4.5.3.1 Initiation Stages 176
4.5.3.2 Hydrogen-Atom Transfers 176
4.6 Pericyclic Reactions 180
4.6.1 Chorismate Mutase 181
4.6.1.1 Models: Catalysis by Antibodies 182
4.6.2 Antibodies Catalyzing the Diels–Alder
Reaction 185
4.6.2.1 Supramolecular Catalysis of the
Diels–Alder Reaction 187
4.6.3 Catalysis by RNA 191

Chapter 5 Design vs. Iterative Methods – Mimicking the Way Nature

Generates Catalysts 195

Introduction 195
5.1 Catalytic Polymers 197
5.1.1 Synzymes 199
5.1.2 Dendrimers 202
5.1.2.1 Peptide Dendrimers 204
5.1.3 Molecular Imprinting 209
Contents xi

5.2 Catalytic Antibodies 212

5.2.1 Other Approaches 214
5.2.2 Proton Transfer from Carbon 216
5.2.3 Conclusions 219
5.3 Nucleic Acids as Catalysts 220
5.3.1 Mechanisms of Nucleic-acid Catalysis 220
5.3.2 Selection as an Alternative to Design Strategies 225
5.3.3 Access to New Catalysts Using SELEX 227
5.3.4 Changing the Catalyst Backbone: DNAzymes 228
5.3.5 Nucleic-Acid Catalysis of Other Reactions 229
5.4 Improving Protein Enzymes 231
5.4.1 Challenges in Exploring Protein Catalysts 231
5.4.2 What Fraction of Diversity Space is Practically
Accessible? 233
5.4.3 Mapping Enzyme Function in the Proteome:
Protein Superfamilies as a Basis for
Understanding Functional Links 236
5.4.3.1 The Enolase Superfamily 236
5.4.3.2 The Alkaline Phosphatase
Superfamily 238
5.4.4 Challenging Chance by Design and
Directed Evolution 243

References 248

Subject Index 266

CHAPTER 1

From Models Through Mimics

to Artificial Enzymes

Enzymes are the all-purpose catalysts that make the Chemistry of Life run
smoothly and efficiently. They do the sorts of things that chemists want to
do, under the mildest, ‘‘greenest’’ conditions – in aqueous solution near
pH 7, at atmospheric pressure and temperatures close to ambient. They are
wonderfully efficient catalysts, capable of handling with ease the most
unreactive compounds present in biological systems, and their reactions,
where necessary, are completely chemo-, regio- and stereoselective. Small
wonder that an understanding of how enzymes work has been an ambition
for generations of researchers in a whole range of disciplines, from pure
enzymology, through almost all of chemistry to X-ray crystallography.
An understanding of the principles of enzyme catalysis is of far more than
academic interest. The industrial use of enzymes is widespread and growing.1
The food industry has always used the enzymes present in various organisms
such as yeasts, but a growing trend is to use isolated enzymes at key stages to
improve quality control. Medical applications have become hugely important,
both in diagnostic testing and directly in therapeutic applications. And
nowhere is the need to understand the fundamental principles more important
than in the development of artificial enzymes, which have far-reaching potential
in the fine chemicals and pharmaceutical industries. Thus, asymmetric synthesis
is a major activity and growth area for organic and pharmaceutical chemistry,
and chiral catalysis the most elegant – and most efficient – way of achieving it.
Enzymes are chiral catalysts par excellence, and natural (wild-type) or specifi-
cally modified enzymes play increasingly important roles.
As proteins, enzymes do, however, have certain practical disadvantages
outside their native organisms: they are often denatured inconveniently fast, by
changes in pH, heat or solvent, and by surfactants and many other chemicals.

From Enzyme Models to Model Enzymes

By Anthony J. Kirby and Florian Hollfelder
r Anthony J. Kirby and Florian Hollfelder 2009
Published by the Royal Society of Chemistry, www.rsc.org

1
2 Chapter 1
And the typical enzyme works best on just one specific substrate, in water, and
at concentrations that are inconveniently low for serious synthesis. Hence the
interest in developing synthetic ‘‘artificial enzymes’’: which can be more robust,
can work in a solvent or solvents of choice; and could in principle be designed
to catalyze a particular reaction, rather than a particular reaction of a specific
substrate. Last but not least, a major advantage of synthetic systems is that they
can in principle be designed to catalyze any reaction of interest, including non-
natural reactions, for which no natural enzymes exist. Successful design in this
context will inevitably be based on developing enzyme models.
Enzymes are far more than just highly evolved catalysts for specific reactions:
they may also have to recognize and respond to molecules other than their
specific substrate and product, as part of the control mechanisms of the cell.
The evolution of artificial enzymes is at a much more primitive stage, with
efficient catalysis the primary, and often the sole, objective. Systems are known
that model various other functions, including potential control mechanisms.
But to be useful as an industrial catalyst an artificial enzyme has no need of
sophisticated built-in feedback control mechanisms or high substrate specifi-
city: a stable molecule that is an efficient catalyst for a key target reaction in a
chemical reactor will not be required to select its substrate from many hundreds
in the same solution, as enzymes routinely must in the cell. So, a rational design
strategy is indeed to consider simply those features of enzymes that are essential
for catalytic efficiency.
In these first two chapters we discuss enzyme mechanisms in rather general
terms, to identify and define these key features. We then go on to discuss the
developing range of enzyme models: by which we mean systems designed to test
basic ideas on enzyme mechanism by reproducing specific, key features of
enzyme reactions; and attempts to develop them into enzyme-like catalysts. We
reserve the term enzyme mimics for the most highly developed enzyme models,
which combine successfully more than one of these key features, and catalyze
reactions by mechanisms that are demonstrably enzyme-like, involving both
binding and catalysis. An enzyme mimic that can do all this, and achieve
turnover at a reasonable rate, deserves to be called an artificial enzyme.

1.1 Introduction to Enzyme Chemistry

Enzymes are proteins. The ‘‘central dogma’’ of biological chemistry underlines
the pivotal role of enzyme catalysis (and highlights a fascinating problem in
biochemical evolution!):

enzymes enzymes enzymes

DNA ! RNA ! Proteins ! Chemistry

Enzyme proteins are made up of one (sometimes more than one) polypeptide
chain (Figure 1.1), each of which is folded into a flexible, more or less unique
active conformation. The preferred 3-dimensional structure is determined by a
complex array of physicochemical interactions between side-chains, main-chain
From Models Through Mimics to Artificial Enzymes 3

R1 H O R3 H
– H2N –
O + O– O etc. Proteins are composed of long
+H N
NH3 2 chains of the 20 naturally occurring
+ L-α-amino-acids, linked by peptide
O H R2 O
(amide) bonds. The primary
sequence of the amino-acids of
enzymes + RNA in the ribosome a natural protein is derived from
the genetic code, and the tightly
controlled synthesis carried out in
R1 H H O R3 H H O Rn H
a complex "machine" called the
N N –
O ribosome, one of the miracles of
NH3 N N
+ molecular biology (and acknowledged
O H R2 H O H Rn-1 H O on the cover of this book).
n
fold

Functional protein

Figure 1.1 Protein structure and biosynthesis.

amide groups and especially solvent water. Whole books and half a dozen
current journals deal specifically with protein chemistry, and the basic ideas are
described in many textbooks. So, only those properties of special relevance to
catalysis will be introduced in this chapter, and discussed in the necessary detail
later in the book. Specific suggestions for further reading are to be found at the
end of each chapter.
The easiest way to a broader understanding of the 3-dimensional structures
of proteins is to spend time on your computer ‘‘playing’’ with real structures.
A good place to start is with the simple-to-use software available at http://
www.umass.edu/microbio/rasmol/ or http://www.pymol.org/). While the
structure of practically any enzyme of special interest is likely to be one of
the many thousands accessible online from the Protein Data Base (http://
www.rcsb.org/pdb/).

1.1.1 Why are Enzymes so Big?

Enzymes have evolved to operate under most of the various environments
natural to living organisms. The most important of these are the cytoplasm –
an aqueous solution containing hundreds of other proteins and small-molecule
metabolites – and the surfaces of membranes of various sorts. So enzymes have
to be ‘‘comfortable’’ in various operating environments, and ‘‘tunable’’ – to work
at different, controlled levels of activity appropriate to the changing requirements
of the system. They must also be capable of catalyzing specific reactions of
specific substrates at rates (based on values of kcat – see Section 1.2 – typically in
the range 1–1000 s1) high enough to support the immediate demands of the
interactive network of local control mechanisms. Substrates range in size from O2
and CO2 to macromolecules, and kcat values between 1–1000 s1 can represent
4 Chapter 1
20
accelerations of up to 10 compared with rates of the corresponding uncatalyzed
reactions at physiological pH in water.
So, an enzyme has to provide a highly sophisticated single-molecule ‘‘reac-
tion vessel,’’ to support such rapid reactions. Not surprisingly, filling and
emptying this ‘‘reaction vessel’’ efficiently poses its own problems, because
when reactions become very fast, simple diffusion processes can become rate
limiting. So, important additional requirements for the active-site ‘‘reaction
vessel’’ are rapid substrate binding and – no less important – rapid release of
product. (This last is no trivial requirement, considering that the product is
typically almost identical to the substrate, give or take the cleavage or for-
mation of a single covalent bond.) Furthermore, most if not all of any water
molecules present in the resting active site have to be removed for the duration
of the reaction process. All this makes the simple picture of a (static) cavity
complementary in structure to the substrate a highly unconvincing proposition.
Fischer’s imaginative lock-and-key principle2 remains a valuable starting point,
but an enzyme must provide specific binding complementarity not just to its
substrate (or substrates), but to all intermediates and transition states on the
reaction pathway.
Thus, the processes involved in catalysis are dynamic, and make complex
demands on the enzyme protein. So, we should not be surprised that all this
should require a large, sometimes a very large, molecule. An additional if less
immediate factor is what might be called ‘‘protein bloat’’. We are all familiar
with the way systems like software applications (or government legislation) that
are continually being improved and extended, can grow rapidly in size and
complexity over just a small number of years. Enzyme evolution is a great deal
slower – but it has been going on for millions of years . . .

1.1.2 Functional Groups Available to Enzymes

What makes one protein different from another is the sequence of amino-acids,
and thus the arrangement of the side-chains Rn (Figure 1.1) in the polypeptide
chain. The chemical reactions involved in enzyme catalysis are implemented by
the functional groups available on the amino-acid side-chains (backbone amide
groups are not usually directly involved). Nine of the 20 naturally occurring
amino-acids carry one of six different reactive groups (seven if phenol and
alcohol OH groups count as different). These are listed in Table 1.1, which also
gives the standard one- and three-letter abbreviations for the amino-acids, and
approximate values for the pKas of the side-chain groups. The pKa value tells us
how much of each ionic form of a group is present at any given pH, for the
amino-acid free in solution. Though it gives only an indication of what might
be the situation in the controlled environment of an active site, where in the
absence of full solvation pKas can be perturbed, sometimes by as much as 4–5
units. (Qualitatively, this perturbation generally favours neutral species,
because ionized forms are stabilized more strongly by hydrogen-bonding
solvation.)
Table 1.1 Amino-acids with side-chains ionizing in the pH region.

From Models Through Mimics to Artificial Enzymes

Amino acid Side-chain functional group Catalytic function of the group

Aspartic acid OH pKa ~ 4

O The COOH group can act as a general acid, but will be
(Asp, D) • • – present near pH 7 only in a specially controlled
O
environment.
O
Glutamic acid • OH pKa ~ 5 • O The carboxylate anion can act as a general base or as a
(Glu, E) – nucleophile.
O O
Histidine • • The imidazole group is about 50% protonated at pH 7,
(His, H) so is the most versatile side-chain group: making
pKa ~ 7
HN + NH
available general acid, general base or nucleophilic
N NH
groups near pH 7.
Cysteine • pKa ~ 9 • The thiolate group is a powerful nucleophile, and more
(Cys, C) SH S– than 1% may be present in a controlled active-site
environment if its pKa is lowered.
Lysine (Lys, + pKa ~ 10 NH2 is a good nucleophile, especially for C¼O, and its
K) • NH3 • NH2 effective pKa can be lowered in a controlled active-site
environment.
Tyrosine • pKa ~ 10 •
An oxyanion is a good nucleophile, especially for
(Tyr, Y) phosphorus.

OH O–
Arginine H The guanidinium group generally stays protonated. It
(Arg, R) N + NH2 pKa ~ 12 N NH2
makes strong, stabilizing double H-bonding interac-
s-Bu •
tions with CO2 and PO2 groups.
NH2 NH2

Serine (Ser, S) OH OH The OH group is not ionized significantly in the pH

Threonine • , • pKa high (13-14) region, but can act as a nucleophile as the proton is
(Thr, T) removed by a general base.

5
Me
6 Chapter 1
The functional groups listed in Table 1.1 are by no means an impressive
selection of reagents, by the standards of the present-day organic or inorganic
chemist. Nor can they be, because they must operate, and have long-term
stability, in water: any strong base, acid or electrophile would immediately be
neutralized by the solvent.

1.2 Principles of Catalysis by Enzymes

Most current thinking about enzyme catalysis is based on Pauling’s original
suggestion that enzymes work by binding and thus selectively stabilizing the
transition states for their reactions. Starting from simple transition-state
theory: consider the interaction between two reacting molecules A and B.
The essential first step is for the two to come together. In the gas phase this
involves a simple collision, but in solution molecules are separated by bulk
solvent, and since each has its own solvation shell, making contact is a more
complicated business. We can allow for this step by introducing into the
reaction pathway the ‘‘encounter complex’’ A . B: without defining it in detail.
Once in contact the molecules can undergo multiple ‘‘collisions’’ within the
encounter complex before either reacting or diffusing apart. Thus, simple
geometrical requirements for reaction, e.g. the directionality of approach of
the reacting centres on the separate molecules, are not generally critical. If the
chemical reaction is faster than diffusional separation, as is typically the case
for many proton-transfer reactions, the diffusion step is rate determining and
the reaction is diffusion controlled. (For examples, see Section 4.1.)

Ka diffusion
A þ B Ð A  B Ð ABz ! ½products Ð products
diffusion

A ball-park estimate of the equilibrium constant for the random association

of small molecules in aqueous solution is Ka B 0.07 M1. Making the interac-
tion between A and B ‘‘sticky’’ – i.e. by the various binding interactions of
molecular recognition – will increase Ka, and thus, other things being equal,
(see Figure 1.4) the overall rate of formation of products. Enzymes work by (i)
binding their own particular substrate, usually very specifically from the hun-
dreds available in solution in the cell, (ii) catalyzing a specific reaction of the
bound molecule; and (iii) finally releasing the product into solution.

1/KM kcat
E + S E.S E.P E + products
(i) (ii) (iii)

This mode of catalysis – whether by an enzyme or a simpler catalyst – is

characterized by ‘‘saturation’’ or ‘‘Michaelis–Menten’’ kinetics: whereby a
limiting rate is reached when all catalyst molecules are ‘‘busy’’ – i.e. binding
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 Linguistics - Workbook
Spring 2023 - School

Prepared by: Teacher Brown

Date: July 28, 2025

Unit 1: Literature review and discussion

Learning Objective 1: Problem-solving strategies and techniques
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Historical development and evolution
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Assessment criteria and rubrics
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 4: Ethical considerations and implications
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Learning Objective 5: Fundamental concepts and principles
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 5: Fundamental concepts and principles
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Learning outcomes and objectives
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 7: Diagram/Chart/Graph]
Practice Problem 7: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Fundamental concepts and principles
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 9: Diagram/Chart/Graph]
Definition: Statistical analysis and interpretation
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 10: Diagram/Chart/Graph]
Summary 2: Learning outcomes and objectives
Example 10: Historical development and evolution
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Interdisciplinary approaches
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 12: Research findings and conclusions
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Fundamental concepts and principles
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 14: Diagram/Chart/Graph]
Remember: Problem-solving strategies and techniques
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 15: Ethical considerations and implications
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 16: Diagram/Chart/Graph]
Key Concept: Critical analysis and evaluation
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 17: Diagram/Chart/Graph]
Key Concept: Historical development and evolution
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 18: Interdisciplinary approaches
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 19: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 20: Diagram/Chart/Graph]
Conclusion 3: Current trends and future directions
Example 20: Problem-solving strategies and techniques
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Historical development and evolution
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 22: Theoretical framework and methodology
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 23: Diagram/Chart/Graph]
Key Concept: Practical applications and examples
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Problem-solving strategies and techniques
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Ethical considerations and implications
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Problem-solving strategies and techniques
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 28: Diagram/Chart/Graph]
Key Concept: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 29: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 30: Diagram/Chart/Graph]
Practice 4: Problem-solving strategies and techniques
Definition: Experimental procedures and results
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Learning outcomes and objectives
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Ethical considerations and implications
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Critical analysis and evaluation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Research findings and conclusions
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 36: Diagram/Chart/Graph]
Definition: Statistical analysis and interpretation
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 37: Diagram/Chart/Graph]
Important: Study tips and learning strategies
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Fundamental concepts and principles
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 39: Diagram/Chart/Graph]
Example 39: Learning outcomes and objectives
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Part 5: Key terms and definitions
Remember: Comparative analysis and synthesis
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 41: Problem-solving strategies and techniques
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 42: Diagram/Chart/Graph]
Example 42: Experimental procedures and results
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Interdisciplinary approaches
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Comparative analysis and synthesis
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Statistical analysis and interpretation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Key terms and definitions
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 47: Interdisciplinary approaches
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Theoretical framework and methodology
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 49: Diagram/Chart/Graph]
Practice Problem 49: Historical development and evolution
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Appendix 6: Literature review and discussion
Key Concept: Interdisciplinary approaches
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 51: Statistical analysis and interpretation
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 52: Diagram/Chart/Graph]
Definition: Assessment criteria and rubrics
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Practical applications and examples
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Best practices and recommendations
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Definition: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 56: Diagram/Chart/Graph]
Practice Problem 56: Experimental procedures and results
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 57: Ethical considerations and implications
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Historical development and evolution
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 59: Diagram/Chart/Graph]
Key Concept: Current trends and future directions
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Methodology 7: Historical development and evolution
Practice Problem 60: Key terms and definitions
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 61: Historical development and evolution
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Research findings and conclusions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 63: Diagram/Chart/Graph]
Example 63: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Assessment criteria and rubrics
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 65: Diagram/Chart/Graph]
Note: Learning outcomes and objectives
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 66: Diagram/Chart/Graph]
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