BIOS INSTANT NOTES

Biochemistry
FOUR TH EDITION

David Hames
Nigel Hooper
 BIOS INSTANT NOTES

Biochemistry
FOURTH EDITION

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 BIOS INSTANT NOTES

Biochemistry
  FOURTH EDITION

David Hames and Nigel Hooper
Faculty of Biological Sciences, University of Leeds

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 Garland Science
Vice President: Denise Schanck
Editor: Elizabeth Owen
Editorial Assistant: Louise Dawnay
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© 2011 by Garland Science, Taylor & Francis Group, LLC

This book contains information obtained from authentic and highly regarded sources.
Reprinted material is quoted with permission, and sources are indicated. A wide variety
of references are listed. Reasonable efforts have been made to publish reliable data and
information, but the author and the publisher cannot assume responsibility for the validity
of all materials or for the consequences of their use. All rights reserved. No part of this
publication may be reproduced, stored in a retrieval system or transmitted in any form or by
any means—graphic, electronic, or mechanical, including photocopying, recording, taping,
or information storage and retrieval systems—without permission of the copyright holder.

ISBN 978-0-4156-0845-9

Library of Congress Cataloging-in-Publication Data

Hames, B. D.
Biochemistry / David Hames and Nigel Hooper. — 4th ed.
p. ; cm. — (BIOS instant notes)
Includes bibliographical references and index.
ISBN 978-0-415-60845-9
1. Biochemistry—Outlines, syllabi, etc. I. Hooper, N. M. II. Title. III.
Series: BIOS instant notes.
[DNLM: 1. Biochemical Phenomena—Outlines. 2. Biochemistry—Outlines.
QU 18.2]
QP518.3.H355 2011
612’.015—dc22 2011004854

Published by Garland Science, Taylor & Francis Group, LLC, an informa business,
270 Madison Avenue, New York NY 10016, USA, and 2 Park Square, Milton Park, Abingdon,
OX14 4RN, UK.

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

Visit our web site at http://www.garlandscience.com

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 Contents

Preface ix

Section A – Cells 1
A1 Prokaryotic cells 1
A2 Eukaryotic cells 5
A3 Cell growth 13
A4 Cell imaging 16
A5 Cell fractionation 22

Section B – Amino acids and proteins 27

B1 Amino acid structure 27
B2 Protein structure and function 35
B3 Myoglobin and hemoglobin 47
B4 Collagen 54
B5 Molecular motors 61
B6 Antibodies 69

Section C – Studying proteins 76

C1 Protein purification 76
C2 Gel electrophoresis 84
C3 Protein sequencing and peptide synthesis 90
C4 Immunodetection 97

Section D – Enzymes 101

D1 Introduction to enzymes 101
D2 Thermodynamics 109
D3 Enzyme kinetics 114
D4 Enzyme inhibition 120
D5 Regulation of enzyme activity 124

Section E – Membranes and cell signaling 132

E1 Membrane lipids 132
E2 Membrane structure 138
E3 Membrane transport: small molecules 147
E4 Membrane transport: macromolecules 153
E5 Signal transduction 158
E6 Nerve function 168

Section F – DNA structure and replication 173

F1 An introduction to DNA 173
F2 Genes and chromosomes 178
F3 DNA replication in bacteria 185
F4 DNA replication in eukaryotes 191

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 vi CONTENTS

Section G – RNA synthesis and processing 195

G1 An introduction to RNA 195
G2 Transcription in prokaryotes 197
G3 Operons 202
G4 Transcription in eukaryotes: an overview 209
G5 Transcription of protein-coding genes in eukaryotes 211
G6 Regulation of transcription by RNA Pol II 216
G7 Processing of eukaryotic pre-mRNA 224
G8 Transcription and processing of ribosomal RNA 236
G9 Transcription and processing of transfer RNA 243

Section H – Protein synthesis 248

H1 The genetic code 248
H2 Translation in prokaryotes 254
H3 Translation in eukaryotes 263
H4 Protein targeting 267
H5 Protein glycosylation 276

Section I – Recombinant DNA technology 281

I1 The power of recombinant DNA approaches 281
I2 Restriction enzymes 284
I3 Nucleic acid hybridization 290
I4 DNA cloning 295
I5 DNA sequencing 300
I6 Polymerase chain reaction 303
I7 Site-directed mutagenesis 308

Section J – Carbohydrate metabolism 314

J1 Monosaccharides and disaccharides 314
J2 Polysaccharides and oligosaccharides 321
J3 Glycolysis 325
J4 Gluconeogenesis 337
J5 Pentose phosphate pathway 346
J6 Glycogen metabolism 350
J7 Control of glycogen metabolism 353

Section K – Lipid metabolism 359

K1 Fatty acid structure and function 359
K2 Fatty acid breakdown 363
K3 Fatty acid synthesis 370
K4 Triacylglycerols 376
K5 Cholesterol 381
K6 Lipoproteins 388

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 CONTENTS vii

Section L – Respiration and energy 392

L1 Citric acid cycle 392
L2 Electron transport and oxidative phosphorylation 398
L3 Photosynthesis 413

Section M – Nitrogen metabolism 424

M1 Nitrogen fixation and assimilation 424
M2 Amino acid metabolism 428
M3 The urea cycle 436
M4 Hemes and chlorophylls 443

Further reading 448

Abbreviations 453
Index 456

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 Preface

It is 5 years since we wrote the third edition of Instant Notes in Biochemistry.

Much has happened in the subject since then; it has truly been an exciting
period for biochemical discovery in so many areas. Not surprisingly therefore
we faced the same desire experienced by textbook authors the world over – to
tell student readers all about the new progress that has been made. However,
the reason we wrote the first edition many years ago was to highlight the core
information that a first-year student needs to know as a firm foundation for
future years of study. This new edition remains true to our original goal but
throughout we have taken the opportunity to significantly update the content.
Our firm belief is that, as with earlier editions, this book will provide a fast way
for student readers to recognize and understand the main facts and concepts –
the bedrock of the subject. For those who wish to know more, in Further Read-
ing we have provided details of some of the excellent mainstream Biochemistry
textbooks that take the reader to the cutting edge of the subject, as well as more
detailed reviews of specific topic areas.
As we remarked in the Preface of the third edition, although this book is aimed
at supporting students primarily in the first and second years of their degree, we
know from personal experience of teaching students that it can rapidly become
a valuable and long-term companion that can be checked whenever there is a
need to refresh one’s memory. Therefore, for some students it becomes a useful
revision aid.
In writing the fourth edition, we have updated all of the core topic areas. The
overall aim was to produce a book that was about the same size as the third edi-
tion, since student (and academic staff) feedback was that its relatively small
size and clear focus is a major strength compared with the very large textbooks
that are generally available.
Finally, a few words directly to the student reader. Like any subject studied at
advanced level, Biochemistry can initially seem a daunting (although intellec-
tually highly rewarding) subject. This book is organized in such a way that you
do not need to read it from cover to cover. You can dip into it in line with your
university coursework, seeking understanding and guidance here and there as
your studies progress. Keep it by you as the dreaded examinations approach;
it will prove to be a quick way to revise – and to reassure yourself that you do
know the fundamental aspects. We hope you will find it as useful as many
earlier students told us they found the earlier editions.
David Hames
Nigel Hooper

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 SECTION A – CELLS

A1 Prokaryotic cells
Key Notes

The origin and All organisms on Earth evolved from a common ancestor.
evolution of cells In the prebiotic era, simple organic molecules formed and
spontaneously polymerized into macromolecules. These
macromolecules then acquired the ability to self-replicate
and catalyze other reactions, before being enclosed within a
membrane to form the first cell. Present-day cells are divided
into three main groups: bacteria, archaea and eukaryotes.
Prokaryotes Prokaryotes are the most abundant organisms on Earth and
fall into two distinct groups, the bacteria (or eubacteria) and
the archaea (or archaebacteria). A prokaryotic cell does not
contain a membrane-bound nucleus.
Prokaryote cell Each prokaryotic cell is surrounded by a plasma membrane.
structure The cell has no subcellular organelles. The deoxyribonucleic
acid (DNA) is condensed within the cytosol to form the
nucleoid.
Bacterial cell walls The peptidoglycan (protein and oligosaccharide) cell wall
protects the prokaryotic cell from mechanical and osmotic
pressure. Some antibiotics, such as penicillin, target enzymes
involved in the synthesis of the cell wall. Gram-positive
bacteria have a thick cell wall surrounding the plasma
membrane, whereas Gram-negative bacteria have a thinner
cell wall and an outer membrane, between which is the
periplasmic space.
Bacterial flagella Some prokaryotes have tail-like flagella. By rotation of their
flagella, bacteria can move through their surrounding media
in response to chemicals (chemotaxis). Bacterial flagella
are made of the protein flagellin that forms a long filament
whose rotation is driven by a flow of protons through the
flagellar motor proteins.
Related topics (A2) Eukaryotic cells (E2) Membrane structure
(B1) Amino acid structure (F2) Genes and chromosomes
(B5) Molecular motors (L2) Electron transport and
(E1) Membrane lipids oxidative phosphorylation

The origin and evolution of cells

Despite the huge variety of living systems, all organisms on Earth are remarkably uni-
form at the molecular level, indicating that they have evolved from a common ancestor.
Life first emerged at least 3.8 billion years ago, although how life originated and how the
first cell came into existence are matters of speculation. Experiments have shown that
simple organic molecules can form and spontaneously polymerize into macromolecules

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 2 SECTION A – CELLS

under the conditions thought to exist in primitive Earth’s atmosphere, the so-called pre-
biotic era. The next critical step was the ability of the macromolecules to self-replicate,
as seen with the present-day nucleic acids, and to catalyze other reactions, as demon-
strated for ribonucleic acid (RNA) (Section G1). The first cell is presumed to have arisen
by the enclosure of the self-replicating RNA in a membrane composed of phospholipids
(Section E1), thus separating the interior of the cell from its external environment. The
encapsulated macromolecules would thus have been maintained as a unit, capable of
self-reproduction and further evolution to give rise to the variety of life forms found on
Earth today. The analysis of the deoxyribonucleic acid (DNA) sequences (Section F1) of
organisms has allowed a possible evolutionary path from a common ancestral cell to the
present-day cells and organisms to be deduced (Figure 1).
The living world therefore has three major divisions or domains: bacteria, archaea, and
eukaryotes (Section A2). The bacteria are the commonly encountered prokaryotes in
soil, water and living in or on larger organisms, and include Escherichia coli (E. coli ) and
the Bacillus species, as well as the cyanobacteria (photosynthetic blue-green algae). The
archaea mainly inhabit unusual environments such as salt brines, hot acid springs, and
the ocean depths, and include the sulfur bacteria and the methanogens, although some
are found in less hostile environments.

Prokaryotes
Prokaryotes are the most numerous and widespread organisms on Earth, and are so clas-
sified because they have no defined membrane-bound nucleus. Prokaryotes comprise
two separate but related groups: the bacteria (or eubacteria) and the archaea (or archae-
bacteria). These two distinct groups of prokaryotes diverged early in the history of life on
Earth (Figure 1).

Prokaryote cell structure

Prokaryotes generally range in size from 0.1 to 10 mm, and have one of three basic shapes:
spherical (cocci), rod-like (bacilli) or helically coiled (spirilla). Like all cells, a prokaryotic

Prokaryotes Eukaryotes

Bacteria Archaea Animals Fungi Plants

Common
ancestor

Figure 1. Evolution of cells.

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 A1 – PROKARYOTIC CELLS 3

cell is bounded by a plasma membrane that completely encloses the cytosol and sepa-
rates the cell from the external environment (Figure 2). The plasma membrane, which
is about 8 nm thick, consists of a lipid bilayer containing proteins (Sections E1 and E2).
Prokaryotes lack the membranous subcellular organelles characteristic of eukaryotes
(Section A2). The aqueous cytosol contains the macromolecules [enzymes, messenger
ribonucleic acid (mRNA), transfer RNA (tRNA) and ribosomes], organic compounds and
ions needed for cellular metabolism. Also within the cytosol is the prokaryotic ‘chromo-
some’ consisting of a single circular molecule of DNA, which is condensed to form a body
known as the nucleoid (Figure 2) (Section F2).

Bacterial cell walls

To protect the cell from mechanical injury and osmotic pressure, most prokaryotes
are surrounded by a rigid 3–25 nm-thick cell wall (Figure 2). The cell wall is composed
of peptidoglycan, a complex of oligosaccharides and proteins. The oligosaccharide
component consists of linear chains of alternating N-acetylglucosamine (GlcNAc) and
N-acetylmuramic acid (NAM) linked b1–4 (Section J1). Attached via an amide bond to the
lactic acid group on NAM is a d-amino acid-containing tetrapeptide. Adjacent parallel
peptidoglycan chains are covalently cross-linked through the tetrapeptide side-chains
by other short peptides. The extensive cross-linking in the peptidoglycan cell wall gives it
its strength and rigidity. The presence of d-amino acids in the peptidoglycan renders the
cell wall resistant to the action of proteases, which act on the more commonly occurring
l-amino acids (Section B1), but provides a unique target for the action of certain antibi-
otics such as penicillin. Penicillin acts by inhibiting the enzyme that forms the covalent
cross-links in the peptidoglycan, thereby weakening the cell wall. The b1–4 glycosidic
linkage between NAM and GlcNAc is susceptible to hydrolysis by the enzyme lysozyme,
which is present in tears, mucus and other body secretions.
Bacteria can be classified as either Gram-positive or Gram-negative depending on
whether they take up the Gram stain. Gram-positive bacteria (e.g. Bacillus polymyxa)
have a thick (25 nm) cell wall surrounding their plasma membrane, whereas Gram-
negative bacteria (e.g. E. coli) have a thinner (3 nm) cell wall and a second outer mem-
brane (Figure 3). In contrast with the plasma membrane, this outer membrane is very

Outer  Periplasmic space Cell wall

membrane
Plasma
membrane

Cytosol
Flagellum
DNA
Nucleoid

Figure 2. Prokaryote cell structure.

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 4 SECTION A – CELLS

(a) (b) Periplasmic Outer

space membrane

Plasma  Peptidoglycan Plasma 

membrane cell wall membrane

Figure 3. Cell wall structure of (a) Gram-positive and (b) Gram-negative bacteria.

permeable to the passage of relatively large molecules (molecular weight >1000 Da) due
to porin proteins, which form pores in the lipid bilayer (Section E3). Between the outer
membrane and the cell wall is the periplasm, a space occupied by proteins secreted from
the cell.

Bacterial flagella
Many bacterial cells have one or more tail-like appendages known as flagella. By rotat-
ing their flagella, bacteria can move through the extracellular medium towards attrac-
tants and away from repellents, so called chemotaxis. Bacterial flagella are different from
eukaryotic cilia and flagella in two ways: 1. each bacterial flagellum is made of the protein
flagellin (53 kDa subunit) as opposed to tubulin (Section B5); and 2. it rotates rather than
bends. An E. coli bacterium has about six flagella that emerge from random positions on
the surface of the cell. Flagella are thin helical filaments, 15 nm in diameter and 10 mm
long. Electron microscopy has revealed that the flagellar filament contains 11 subunits
in two helical turns, which, when viewed end-on, has the appearance of an 11-bladed
propeller with a hollow central core. Flagella grow by the addition of new flagellin sub-
units to the end away from the cell, with the new subunits diffusing through the central
core. At its base, situated in the plasma membrane, is the flagellar motor, an intricate
assembly of 40 or so proteins. The rotation of the flagella is driven by a flow of protons
through certain proteins of the flagellar motor. A similar proton-driven motor is found
in the F0F1-adenosine triphosphatase (ATPase) that synthesizes adenosine triphosphate
(ATP) (Section L2).

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