Biochemistry 6th ed j berg, j tymocsko, l stryer (freeman, 2006) 1

Brief Contents Part I THE MOLECULAR DESIGN OF LIFE 1 Biochemistry: An Evolving Science 1 2 Protein Composition and Structure 25 3 Exploring Proteins and Proteomes 65 4 DNA, RNA, and

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Berg, Jeremy Mark

Biochemistry / Jeremy M Berg, John L Tymoczko, Lubert Stryer 6th ed

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To our teachers and our students

About the Authors

JEREMY M BERG received hi s B.S and M S de g ree s in C hemi s tr y from

S tanford ( wher e he did re sea r c h with K e ith Hodg so n and Lubert S tr yer) and his Ph.D in c h e mistr y from Harvard with Ri c hard Holm He then

co mplet e d a p os tdo c toral fellow s hip with Ca rl P a b o in Biophysics at John s

H o pkins University Sc hool of Medicine He was an Assistant Professor in

the Department of Chemistry at Johns Hopkin s from 1986 to 1990 He then moved to Johns Hopkins University School of M e di c ine as Profes so r

and Director of the Department of Biophy sics and Biophysical Chemistry, where he remained until 2003 In 2003, he became the Director of the

National In stit ute of General Medical Sciences at the National Institutes

of Health He is recipient of the Ameri ca n C hemical Society Award in Pure C hemistry (1994 ), the Eli Lilly Award for Fundamental Resear c h in Biological Chemistry ( 1995 ), the Maryland Outstanding Young Scientist

o f the Year ( 1995), and the Harrison Howe Award (1997 ) While at Johns

Hopkins, he received the W Barry Wood Teaching Award (se lected by medical students as award recipient ), the Graduate Student Teaching

Award, and the Professor' s Teaching Award for the Preclini c al Sciences

H e is coa uthor, with Ste phen Lippard, of the textbook Principles of Bioinorganic Chemistry

JOHN L TYMOCZKO is Towsley Professor of Biology at Carleton College,

where he has taught since 1976 He currently teaches Biochemistry, Biochemistry Laboratory, Oncogenes and the Mole c ular Biology of Cancer, and Exer c ise Biochemistry and coteaches an introdu c tory co urse,

Energy Flow in Biological Systems Professor Tymoczko received his

B A from the University of Chicago in 1970 and his Ph.D in Biochemistry

from the University of Chicago with Shutsung Liao at the B e n May Institute for Cancer Research He then had a po st do c toral po s ition with Hewson Swift of the Department of Biology at the University of Chicago

Th e focus of his research has been on steroid receptors , ribonucleoprotein particles , and proteolytic proces s ing enz y mes

LUBERT STRYER is Winzer Professor of Cell Biology, Emeritus, in the

School of Medicine and Profe sso r of Neurobiology, Emeritus, at Stanford University, where he has been on the faculty since 1976 He re ce ived his

M D from Harvard Medical School Profes so r Stryer has received many awards for his research on the interplay of light and life, including the

Eli Lilly Award for Fundamental Resear c h in Biologi c al Chemistry and

the Distinguished Inventor s Award of the Intellectual Property Owners' Association He was e lected to the National Academy of Sciences in 19 84

He c urrentl y c hair s the Scien tific Advisory Board s of two biotechnology

companies Affymax, Inc , and Senomyx, Inc and se rves on the Board

of th e McKnight Endowment Fund for Neuroscience Th e publi ca tion

of his first edition o f Biochemistry in 1975 transformed th e teac hing of

bi oc h e mi stry

PREFACE

h e m o r e w e l e arn , th e m o r e w e di scover conn ec tio n s t h read i ng t h ro u g h o ur

bi oc h e mi c al w orld In wri t in g th e s ixth e di t i o n , we ha ve m ad e eve r y e ff or t

to pr ese nt th ese co nn ec ti o n s in a way that will hel p f ir s t - tim e s tud e nt s o f bi o

-c h e mi s tr y und e r s tand th e s ubj ec t and h o w v er y releva n t i t i s t o t h e ir li ves

Emphasis on Physiological Relevance

Bi oc h e mi s tr y i s r e turnin g t o it s roo t s t o r e n e w the study of it s role in phy s i o l ogy, wi t h

th e t oo l s of m o l ec ul a r bi o l ogy a nd t h e inf o rm a tion gain e d f rom ge n e se qu e n c i n g

in h a nd In t h e s i x th e diti o n, we e mph as i ze th a t an und e r s tandin g o f bi oc h e mi c al

p athw ays i s th e und e rpinnin g f o r a n under s tandin g of phy s iol og i ca l sys t e m s

Bi oc h e mi c al pathwa ys mak e m o r e se n s e t o s tudent s wh e n they und e r s t a n d h ow

th ese p a th w a ys re l ate t o th e ph ys i o l ogy o f famili a r a c ti v iti es su c h a s di ges ti o n , r esp i

-r ati o n, and exe r c i se In thi s e diti o n, p a rti c ularl y in th e chapter s on m e t a b o li s m, we

ha ve tak e n seve ral s t e p s t o e n s ur e that s tudent s have a v iew of th e bi gge r pi c tur e :

• Di sc u s si o n s of m e tab o li c r eg ul a ti o n e mpha s i ze th e everyday conditions t hat

d e t e rmin e r eg ul a ti o n : exe r c i se ve r s u s r es t; f e d v e r s u s f as ting

• Ne w pathway-integration figures s how h o w multiple pathwa ys wo rk

t oge th e r und e r a s p e cifi c co nditi o n, s uch as durin g a fa st

• Mor e physiologically relevant examples hav e b ee n add e d thr o u g h o u t t h e b oo k

Thi s ph ys i o l og i c al p e r s p ec ti ve is a l so e vid e n t i n th e n e w c h a pt e r o n d ru g d e ve l

-o pm e nt Th e u se o f a f o r e i g n co mp o und t o inhibit a s p eci f ic e nz y m e so m et im e s ha s

s urpri s in g ph ys i olog i ca l co n se qu e n ces t hat r e v e al n ew p h ysio l og i ca l prin cip l es

F A S TIN G or D IAB ETES

2 F o rmat i o n o f ke t one bod i es , C ha p t er 22

3 G l u c oneog e n esis, Cha pt e r 1 6

4 Ket one bod i es 7 a c e ty l CcA C h a pt er 22

FAT CELL

Tr ia cy glycerol

Fig ur e 22 21 P athway Integration : Live r

supplies ketone bodies to the peripheral

t i ssues D ur i ng fa s tin g o r in untr ea t e d

di abe t ics th e l iver conv e rt s f atty aci d s into ket o ne bo die s whi c h are a f ue l so urc e fo r

a n umb e r o f ti ssues K e t o n e b o di es a r e t h e

,

v i Prefa ce

A Molecular Evolutionary Perspective

Evo lutionar y p e r spect i ves great l y e nabl e a nd e nh a n ce

the s tudy o f bio c h em i s try A s Theod os iu s Do b z h a n s k y noted, "nothing in biolo gy make s se n se exce pt in the light o f evo lution " In the course of evo luti on, mutati o n s

a lt e r e d many prot ei n s and biochemi c al m o tif s so that

they perform di ffe r e nt fun c tions while maintaining their

co r e bio c h e mi ca l e l eme nt s By exa mining relat e d pro tein s , w e hi g hli g ht esse nti al c h e mi ca l f eat ur es as well

-as th e s p ec ialization n ecessa r y for particular function s

The tracks of evo lution are clear from th e a naly sis of

ge n e a n d prot e in se qu e n ces

As seq u e n ce ana l ysis becomes more important,

the field of biochemistry is s hifting from a science perfo rm ed a lm ost e ntirel y in the l aboratory to one

that ma y a l so be ex plored through co mput e rs , by

u s ing inf or mati o n ga th e r e d from genom i cs and prot eo mi cs Thi s s hi ft i s manif est in th e c urr e nt e di - tion a nd ca n be see n most clea rl y in C h apter 6,

" E xploring E v o luti o n and Bi oinformatics," which dev e l ops t h e co n ce ptual ba s i s for co mparing protein

a nd nucl e i c ac id se qu e n ces Protein co mp a ri so n s a r e a

frequent so ur ce of in s ight throughout th e book,

espe-c iall y for illuminat in g r e lation s b etwee n st ru ct ur e and fun c ti o n

New Chapters : Hemoglobin and Drug Development

Two n ew c hapter s illu s trate t h e r e l ation between

st ru c ture a nd fun c ti o n by using a classic exa mpl e and

a co nt e mp o rar y o n e

Chapter 7 : Hemoglobin : Portra i t of a Protein in Action

Thi s clas s i c ex ampl e, u sed to convey th e relation

b e tw ee n s tru c tur e and function, returns in an ex panded treatment New in s i g ht s includ e :

• Oxygen transport during r es t and d urin g exe rci se

• Th e phy sio l ogy of oxyge n and CO2 transport

• Th e mol ec ular ba s i s of s i ck l e-cell anemia and tha l ass e mia

• Balan c ing th e production of a and 13 chains

• New l y dis cove r e d globins

Chapter 15 : Drug Development Knowledg e of bio

-c hemi ca l pathways i s k ey to the d eve lopm e nt of n ew

drug s s u c h a s Lipitor, Viagra, and Vioxx In thi s n ew

c hapt e r , pl e ntifu l case s tudies illu s trate :

• How drug s r e lat e to ot h er topics in the book

-kineti cs , enzyme inhibitor s, membran e r ece ptor s, metaboli c regulation, lipid sy nth esis, and s i g nal transdu c tion

• How th e body's d e fen ses r es pond to foreign

co mpound s , espec iall y the defenses provided by the biochemical pathway s of xenobiotic metabolism

• The importan ce o f admini s tration, di s tribu t i on,

metaboli s m , exc r e ti o n (AD ME ) , and toxi c ology

from medi c al sc i e n ce to the already abundant

se l ec ti o n of s u c h exa mple s ( indi c ated by the

i c on above ) ( For a full li s t see p x ) N e w topics includ e:

• Diseases of pr o tein mi sfo ldin g ( C hapter 2)

• Human gene therapy (C hapter 5)

• Aggregan and osteoa rthriti s (C hapt e r 11 )

• T h e u se o f e rythrop o i eti n ( EPO ) to tTeat an e mi a and

it s abu se b y athlete s (C hapt e r 11 )

• The u se of m o n oclo nal antibodie s to target epidermal - growth - fa c tor receptors in the treatment

of co l on and breast c an ce r s (C hapt e r] 4 )

• Ro l e of exercise in building defenses against

• Much e nhan ce d discussion of gout (C hapter 25)

• Folic a c id and spi na bifid a (C hapter 2 5 )

• Typ e II dia b e tes (C h a pt er 2 7 )

• Tumor s uppr essor ge n es and p 53 (C hapt e r 2H)

• C hem o therapy targeting DNA repair pathway s

( Chapt e r 28)

• Diseases of d efec tiv e R NA sp li c in g, including

thala ssem i as a nd retiniti s pi gme nto sa (C hapt er 29)

• Inna te immunity ( Chapter 33)

Recent Advances

The s ixth e diti o n ha s been thoroughly updat ed

through-o ut , includin g new dis c u ss ion s of the following recent

advan c es:

• The nucleation condensation model of protein

folding ( Chapter 2)

• Us in g MALDI-TOF mass spectrometry to identify

co mpon e nts o flarg e protein co mpl exes (C hapter 3)

• Update on th e human genome project (C hapter 5)

• Comparative genomics (C h apter 5)

• Gen e di sr upti o n b y RNA interference (C hapt e r 5 )

• Us ing BLAST searches (C hapt er 6)

• Lipid rafts (C hapt e r 12 )

• Mechanisms of action of several types of membrane

c hann e l s and pumps s u c h as the acetylcholine

• DNA polymerase co mponents (C h apte r 2S)

• Th e trombone model of DNA elongation (Chapter 28)

• Promoter structure in e ukar yotes (C hapter 29)

• Transcription initiation in e ukar yotes

Figure 14 20 Insulin signaling The binding

of i n sulin to its receptor leads to a se ri es

of phosphorylations, resulting in the act ivati on of the kinase Aktl Activated Akt1 diffuses thr o u g h out the cell to

continue the Signal-transduction pathway

vii i Preface

Visualizing Molecular Structure

As in the fifth e diti o n, all mole c ular st ru c ture s hav e

b ee n se l ecte d and r e nd e r e d b y o n e o f us , J ere m y Berg

T h e s ixth e diti o n include s new t oo l s to help students

r ea d and und ersta nd mol ec ular s tru c tures :

• A mol ec ular model " primer" ex plain s th e different

• A greater var i e t y of types of mol ec ular s tru c tur es

a r e r ep r esented , in clud in g clea r e r renderings of

membran e proteins

• For m ost molecular models, the name of the file

from the Protein Data Bank is given at th e e nd

o f th e figure l ege nd This f il e nam e (a l so known

a s a PDB ID) allows t h e reader easy access to

th e fil e used in ge n era ting th e st ru c ture from the

Protein Data Bank W e b s it e ( http ://w ww r cs b

org/pdb/ ) At thi s s it e, a va riet y o f to o l s for

v isuali z in g and analyzing the s tru ct ur e are

avai labl e

(A)

Iron atom

(8)

• Living Figures for most m o l ec ular st ru c ture s now

appear on the Web s ite in J m o l t o allow s tud e nt s

to rotate 3- D mole c ul es and vi e w alternative

• Mechanism problems a s k st udent s to s ugg es t or

elaborate a chemic a l mechani s m

• Data interpretation problems a s k questions about

a set of data provid e d in tabu l ated o r graphic form

The se problems give students a sense of h o w

sc ientific conclusions are r eac hed

• Chapter integration problems requir e st ud e nts to

u se information from severa l c hapter s to r eac h a

so lution These problem s reinforce a student's

awar e n es s of the interconn ecte dne ss of th e different

aspects o f bio c hemi s try

Brief so lution s to these problems are pre se nted at the end of the book ; expanded so lutions are available in the accompanying Student Co mpan ion

Heme

~ Figur e 2.48 Three-dimensional structure of m y oglobin {Al A r ibbo n diagram s h ows

that the protein consists largely o f a hel i ces {Bl A space-filling model i n the sa m e orientation shows how ti ghtly packe d the fol de d protein is Notice that the heme group is

nestled into a crevice in t he com p act p rotein wi th on l y an edge exposed One hel i x is blue

to allow compa r ison of the two structural d ep ictions [ Dra wn from lA6 N.pdb.]

Molecular Evolution

This i co n signa l s the start of many discussions that hi ghlight protein commonalit i es or other molecular evolutionary insights that provide a framework to help students organize information

Why thi s se t of 20 amino a c ids ' ( p 33)

I s the ge net ic code univer s al? ( p 126 )

Many exons e n co de prote in domains ( p ] 2 8)

Fetal h e moglobin s ( p 1 92)

Additiona l g lob in s ( p 197 )

Ca talyti c triad s in hydrolyti c enzymes ( p 24~)

iV l ajor classes o f peptide - c l e aving enzymes ( p 2 51 )

Zinc-based a ctive s ites in c arboni c anhydrases ( p 2 58)

A co mm o n cata l ytic core i n lype II restri c tion enzymes ( p 26 6 )

P - I oo p NTPase d o main s ( p 270)

A co mm o n c atalyti c co r e in pro tei n kinas es ( p 2 88 )

Why might human blo o d type s differ ? ( p 3] 5)

Ar c haeal membrane s ( p 331)

P-typ e ATPas es (p 354 and p 3:;8)

ATP - binding c a sset te domains ( p 35~)

Using se qu e n ce co mp a rison s to und ers t a nd Na t and Ca 2 I

c hannel s ( p 366)

S mall G protein s ( p 398)

E vo l uti o n o f metab o lic p a thway s ( p 429 )

Why is g lu cose a prominent fuel ? ( p 43:; )

A co mm on bindin g site in dehydro ge na ses ( p 448 )

T h e maj or facilitator ( MF ) s uperfamily of tran s porter s ( p 457)

I sozymic forms o f la c tate deh y drogenase (p 469)

Evolutionary relationship of g l yco l ysis and gluconeogenesis

( p 46 9)

Dec a rb o xy la ti o n of ,, - keto g lutarat e and pyruvate (p 48:;)

Evolution of s u cc inyl eoA sy nthetase ( p 487)

Evolutionary hi s tory of the citri c acid cy cl e ( p 495)

Endosymbiotic o rigin s of mitochondria ( p 50 4 )

C on se rvation of cytoc hrom e c st ru ct ure ( p 52 0)

C omm o n f e ature s of ATP sy ntha se and G prot ei n s ( p 527)

Related un c oupling pr o tein s (p 533)

Evolution of c hloropla s t s (p 543)

Evolutionary origin s of photo sy nthesi s ( p 56 0 )

Evo lu t ion o f the C4 pathway ( p 576)

Incr eas ing sophistication of g lyco ge n phosphorylase r eg ulation (p 604 )

The ", - amyla se family ( p 606 )

A re c urrin g motif in the a c t i vation of carbo xyl gro up s ( p 623)

Prokaryoti c co unterpart s of t he ubiquitin p at h way and the

pro teasome ( p 655 )

A famil y of pyridoxal - depend e nt enzymes ( p 660)

Evolution of the urea cycle ( p 664)

Th e P - I oo p NT P ase domain in nitr oge na se ( p 6R 2)

Aspartat e aminotran sferase, prototype of PLP - d epen d e nt e nzyme s

( p 687)

Feedba c k inhibiti o n ( p 698)

Recurring s tep s in purin e rin g syn the sis ( p 7 1 5)

lZibonucleotide redu c t ases ( p 720)

Incr e a se in urate le ve l s during primate e v ol ution ( p 726)

The cy to c hrome P 450 superfamily ( p 752)

D N A p o l yme ra s e s (p 794 )

Helicase s ( p 798)

Thymine and fide l ity of ge n e ti c message ( p ~09)

Evolutionary relation s hip of recombinases and topo i some r ases

( p 8 14 )

Evolut ion of s pli ceoso m e-ca tal yze d s pli c in g ( p 850)

C lasses of aminoacyl-tRNA sy nth etases ( p 865)

Composition of t h e pr i morda l r iboso m e ( p R69)

Hom o l og ous G protein s ( p 877)

A family of protein s with co mm o n li ga nd binding domains ( p 899)

Ind e pend e nt e voluti o n of DNA - binding s ite s of re g u l at o ry protein s ( p 900)

CpG i s lands ( p 907)

Iron resp o nse elements ( p 9 1 6)

The o dor a nt rece pto r fa m i ly (p 923)

Photoreceptor evo lutio n ( p 936 )

The immunoglobulin fo l d ( p 952)

Relation s hip of actin to hexokina se and prokaryo t i c protein s ( p 9Xb)

Tubulins in P-loop NTPase fami l y (p 990)

Clinical Applications

" T A dditi hi s i co o n n a l s , i g bri nal efe s th r e s cl ini tart ca l o co f a rrelati cl ini ca o n l s appea a p plic ati r in th o n m th e te e x t t as ext

ap p ro pri a t e

Diseases of protein misfoldin g ( p 53)

Disea se due to fa il ure of prot ei n modifi c a t ion ( p 57 )

Anti gen dete c tion with E Li CiA ( p 87)

Va so pre ss in def i ciency ( p YO )

Carboni c anhydrase and os t eo petro s i s ( p 25 4 )

Use uf i!:)Ozym es to dia g no~e ti ss u e clama ge ( p 28 3 )

Emphy se ma ( p 2 9 2)

Thrombose s preventi o n ( p 295)

H emophi l ia ( p 2 97 )

Regu l at i on of blo o d clott i ng ( p 297)

A gg re g an and osteoart hr iti s ( p 313)

Cli ni ca l u ses oflipusumes (p 335)

Aspirin and ibuprof e n ( p 339)

Di g ital i s and congestive h eart fa ilure ( p 357)

Mul t idru g resi s tan ce ( p 358)

Signal - tran s duction pathw ays a nd cance r ( p 400 )

Monoclona l antib od i es as a nti ca n cer Jru gs ( p 401)

P rotei n kina se i n h ib i tors a s a nt i cancer drugs ( p 40 1 )

Chole ra and w h ooping c ough ( p 401 )

V i tam i n def i c i e n c ie s ( p 4 23)

L actose in t o l eran ce ( p 451)

Galactose tox i c i ty ( p 45 1 )

Can ce r a n d g ly co ly sis ( p 457 )

Phospha t a se defi ci en c y and l act i c acidos i s ( p 4n )

H eribe ri and poi s oning by mer c ury and a r senic ( p 494 )

M it ochond r ial di s eas es ( p 534)

H emo l y t i c a n emia ( p 586)

Glucose 6-phosp h ate dehydrogenase def i c i e n cy ( p 587)

G ly coge n - s tora ge d i se a ses ( p 61 1)

Ca rni t ine d ef i c ienc y ( p 624)

Ze llw eger sy nJrom e (p 630)

Diabetic k e to s i s ( p 6.B)

Use of fatty ac id syn th ase inh ibito r s as Jru gs ( p 640)

Effects of aspirin o n s i g nal i ng pat h way s (p 644)

x

Diseases of alte r ed u biquination (p 653)

Protein degrad a t i o n and th e inflammatory r esponse ( p 664 )

I n h e r ited defects o f the urea cy cle ( hyperamm o n e m i a) ( p 664)

i nborn errors of amino acid degrauation ( p 672 )

H i g h homo cys teine l eve ls and vascu l ar disea se (p 693)

I nherited disor d ers of porp h yr i n metabolism ( p 70 4 )

An t i c ancer drug s t h at block the synt h es i s of t h ymidy l ate ( p 722)

Adenos i ne deaminase and Severe co mb i ned immunodefic i e n cy

(S ClD ) ( p 725)

Cout (p 726)

Lesch - Nyhan syn d rome ( p 726)

Foli c acid and spi n a bifida ( p 727)

Respiratory d i s t ress syn d rome and Tay-Sacl1S disease ( p 738)

D i agnostic use of b l ood cho l estero l levels ( p 74 5)

Hyper c h o l este r em i a and athe r oscleros i s ( p 7 4 7)

C l inical m a n ageme n t of cho l esterol l evels ( p 748 )

D e f ec t ive r epa ir of D NA an d ca n cer ( p 8 1 0)

D etec t io n of ca r ci n ogens ( Ame s tes t ) ( p 81 1 )

Tools and Techniques

The s ixth ed ition of Biochemistry offer s three c hapt e r s that pre se nt the tools

and techniques of bio c hemi st r y: "Exploring Prot e ins and Proteomes"

( C hapt e r 3), "Exp loring Genes and Genom es" (C hapt e r 4 ) , and "Exp loring

Evolution a nd Bioinformatics " ( Chapter 6 ) Additional ex p erime ntal tech

-nique s are presented throughout the book, as appropriate

Exploring Proteins and Proteomes (Chapter 3)

Protein purifi c ation ( p 67 )

Oiff e r e ntial ce ntrifu g ati o n ( p 67 )

Saltin g out ( p 68)

Dial ysis ( p 69)

Gel-fi ltra ti o n c hr o mato g raph y ( p 69 )

I o n - exchange c hromato grap h y (p 69 )

Affinity c hromat ogra ph y ( p 7 0 )

H igh - pre ss u re li quid c hromat og raph y ( p 7 1 )

Ge l electrophore s i s ( p 71)

I soelec tri c f oc u s in g ( p 73 )

Two - dim e n s ion al ele c tr o ph o re s i s ( p 74 )

Qualitati ve and quant itat ive evaluation of protein purifi ca ti on (p 74 )

Examinin g expression l eve l s (ge n e c hip s) ( p 1 5 1 )

C reating cDNA libraries ( p 152 ) Introdu cing genes i nt o e ukar y otes ( p 154)

Tran sg en i c anim a l s ( p 1 55 ) Gene di s rupti on ( p 155)

Gene di s ruption by R NA in t erfe r e n ce ( p 1 5 7 )

Tumor - indu ci ng pla s m i d s ( p 15 7)

Exploring Genes (other chapters)

D e nsity - g radi e nt equi li brium ~ ~dimentation ( p 113 )

footprinting t ec hniqu e f o r i solati n g and c hara c t e rizin g

promoter s ites ( p R24)

Chromat in immun op r ecip itati o n (C h II' ) ( p 906)

Produ c ti on of p o lyclona l antibodie s ( p 84 )

Produ c ti o n of m o nocl o nal antibodie s ( p 85)

Enz y me - link e d immun oso rb et a ss ay ( ELI SA) ( p 87 )

Flu o re scence mi croscopy ( p 89)

Green flu o r escent protein as a marker ( p 89 )

[mrnun o el ec tron rni c ro sco p y ( p 89)

Automat ed so li d-p ha se peptide sy n t h es i s ( p 90 )

MALDI - T O r m ass s pe ctro m etry ( p 93)

Prote o mi c anal ysis b y ma ~~ ~ pectrometry ( p 94)

X - ray c ry s tall og raph y ( p 96)

Nuclear ma g n e ti c r eso n a n c e s p ect ros co py ( p ~8 )

NOESY s pe c tro sco py ( p 99 )

Exploring Proteins (other chapters)

Ka s i s of flu oresce n ce in g reen flu o r esce nt pro t ei n ( p 58 )

Tim e - r eso l ved c r ystallog raph y ( p 2 1.1 )

Usi n g flu o r esce n ce s p ec t roscopy to a n a l yze e n zy m e s ub s trat e in ·

t e ra c ti o n s ( p 2 1 3 )

U sin g irr eve r s i b l e inhibit or t o map the active s ite ( p 228)

Enzyme s tudi es with cata l y ti c antibodies ( p 232 )

Exploring Genes and Genomes (Chapter 5)

R es tri c tion - e nzym e a n a l ys i s ( pp 135 - 1 37)

So uth e rn a nd Nort h e rn blotting te c hniyue s ( p 137)

S an ger did eoxy m et h o d of DNA seq uencin g ( p 1 38)

So lid - p h a se sy nth es i s of nucleic a c id s ( p 139 )

P oly m e ra se c hain r eac ti on ( P C R ) ( p 140 )

Re co mbinant DNA t ec hn o l ogy ( pp 1 42 - 1 59) ,

S equence-compari so n m et h o d s ( p 166 )

Seq uen ce-a li gn m e n t method s ( p 1 66 )

Es timatin g th e s t a tisti ca l s i g nifi c an ce of ali g nment s

(by s hufflin g) ( p 168 )

S ub stit uti on mat ri ces ( p 168 ) Performing a BLA ST databa e searc h ( p 1 71 )

Seq uen ce templat es ( p 1 7 4 )

I l e te c tin g rep e ated m o tif s ( p 1 74)

Mappin g secon d a r y str u c tur es throu g h R N A se quen c e

Use of lip oso me s to investi g ate m embrane permeabi lit y ( p 334)

Use of hydropath y p lo t s t o l oca te tran s membrane heli c e s ( p 3 4 0)

Flu o re sce n ce r ecove ry af t e r ph o t o bl eac hin g (FRAP ) for m e a s urin g

lateral diffu s ion in m e mbr a n es ( p 142)

Patch · clamp technique for m e a s ur i n g c hann e l a c tivit y ( p 363)

Mea s uremen t o f r e d o x potential ( p 506)

Fun ct ional ma g n e ti c re so nan ce imaging ( fM RI ) ( p 926 )

Animated Techniques : Animated exp lanat io n s of

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pr ote in s a r e ava ilabl e at www w h fre e man c om / stryer

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Living Figures

T hi s ico n i d e ntifi es m o l ec ular stru c ture s that a r e a vail a b l e in r o tat a b l e

J m o l f o rm a t o n t h e co mp a ni o n W e b s it e: www w h f r ee m a n co m / st r yer

Structu r e dictates func l io n : a p r ote i n ~u r roun d ing DNA Figure 2 1

Co nformatia n al change i n l ac t oferrin Figure 2.3

Ferr i t i n, a l a r ge l y a - hel i ca l p r o t ein F i gu re 2.33

A fa tt y-ac i d b i nding protei n r i c h in i3 s h eets F igu r e 2 4 0

Loops on an a n tibody prote in su r face F i gure 2.42

An a - h e li cal coiled co il Figu r e 2.43

Heptad r epeals in a c oi l ed coi l p r ote i n Figu r e 2.44

Three - dimen s i o nal s tructu r e of myoglobin F i gure 2 4 8 Distribution of amino ac i ds i n myoglobin Figure 2.49

" l n s iJ t:: out" ami n o acid distri bution in perin Figure 2.50

The helix - turn - he l ix motif Figure 2.51 Protein domain s of the ce ll s urface protein C ]) 4 Figu r e 2.52

Quate rn ary s t r u c tur e of t h e era prote i n of bac t er i ophage

"-Figure 2.53

The Q' 2 f3 2 tetramer of human hemog l obin Figure 2.54

Alte r n a tive co n formation s of a peptide sequence Fig ure 2.60

C h em i ca l rear r angeme n t in gree n fl uorescent pro t e i n (G F P)

Figu r e 2.68

Repeating mot i fs in c almoduli n F i g u re 3.25

I mmu n oglob u lin G antibody F i gure 3.27

A n tige n {lysazyme )-a nt i body i n t eract i ons Figure 3.28 Watso n - Crick model of double- h elical DNA Figure 4.11 RNA po l ymera se Figure 4.24

A pro t e i n with no natu r a l counte r part Figure 5.22

Ribonuclease from cows and human being s Figu r e 6.1

Ang i oge n i n Figure 6 2

I - T u m a n he m oglobin (cr c h ain ), human myoglobin, and l up i ne

leg h emog l obin Figure 6 14

Ac t in a nd the l a r ge frag m e n t of h eat s h ock pro t ein 70 (H sp-70 )

Figure 6.15

Chymo t ry p sin and s u btilisin Figure 6 1 8

Myog l obi n Figure 7.1

Quate rn a r y struct u ral c h a n ges in h e m og l ob i n o n o x y gen b in d i n g

F ig u re 7.10

~1ode o f b in di n g of2,3- BP G t a hum an deoxy h cmog l ob in Figure 7.16

[l e mog l obin S Figure 7 25

~tablizing free a - hemog l ob in F i g ur e 7.27

A co mplex of lhe enzyme cytoc hr ome P 450 and it s subs tr a t e

c amphor Figure 8.5

Lysozyme w i th severa l co mpo n e n ts o f t h e ac t ive s it e F igu r e 8.7

Chymo t rypsin F ig ure 9.6 Tryp s in and chymotrypsin Figure 9 12

Carboxypeptidase II F ig ure 9.15

Three classes o[ pro l eases and their act i ve sites Figu r e 9.l7

H I V pro t ea se and its binding pocket Figure 9.19

H I V protea se, a dimeric a s partyl protea se Figure 9.2 1

H uman carbon i c an h ydrase I r and it s zinc s i te Figu r e 9.22

)' - Carbon i c a nh ydrase Figure 9.31

Eco R V embr ac ing a c og n ate DNA m olec u le Figu r e 9.38

H y d roge n bo n ding in te r actio n s b etwee n E co R V e nd o nu clease a n d

i ts DNA s ub stra t e Fi gu r e 9.39

XII

Nonspe c i fi c and cognate DNA w i t h in E co R V e nd o n uclea se

Fig u re 9.41

A conse r ve d st ru c tu ra l core i n type II res tri ct i o n e n zy m es Fig ur e 9.44

Adeny l ate k i nase a n d g u any la te kinase Figu r e 9.46

T h e core domain af N M P kinases Figure 9.47

Co n fo r mat i ona l c h anges in adeny l ate ki n ase Fig u re 9.51

Th r ee prote in s c ontai ni ng P -I oop NT P ase d omai n s F i gu r e 9.52

A TCa.e Figure 10.6

Th e active s i te af ATCase Figure 10.8

Prote i n kinase A bo un d to an i nh ih i tor Figure lO.l8

Conformation s of c h ymot r y psinoge n (r ed ) a n d c h ymot r yp s i n

( bl ue) Figure 1 0 22

I nte r a ct io n of t r yps in w i th i t s inhib i to r F i gure 10 2 4

A fi br i !loge n mo l ecu l e Figure 10.27 The calc i um - b i n di ng r egion of prothrombin Figure 10.32

Oligosaccha r ides attache d t o e r ythropo i et i n Figure 11 2 1

Struc tur e of a C - t ype c ar bo h yd r ate - binding do m a i n fr om a n

a ni ma l lec ti n F igure 11.26 Bacterio rh odopsin F i g u re 12 1 8

B acter i al po r i n ( f ro m W lO d opse u durnuna s b l astica ) F i g u re 12.20

Attach m e n t of prostag l an di n H2 synt h ase· ' to th e m e mbr a n e

A l actose perme a se w ith a bo und l actose a n a l og Figure 13.11

The p o ta ss ium c ha nn el Figure 13.17

A vo l t age - gate d po tassi um c h a n nel F i g ur e 13 22

Th e ac etylch o li ne r ece p to r Fi g ur e 13.27

A q ua p ori n F i g ur e 13.33

7 T M r ecep t or F i gure 14.4

A h e t ero tri me r ic G pro t ei n Figure 1 4.6

A d e n y l a t e cy cl ase act i vat i o n F i g u re 14 7

I n d u ced fi t i n hexo k inase Fi gure 1 6.3

T ri ose p h osp h a t e iso m e r ase Figu r e 16.4

G l yce r alde h y d e , - phos ph ate d e h ydroge n ase F igu r e 16 6

)!AD+ -bi n di n g reg i o n i n de h y d roge n ases F i g u r e 1 6 12

P h osphofructoki n ase F ig ur e 16 1 5

Bi oti n -binding d omai n o f py ru va t e ca rb o x yl a se F igure 1 6.2 4

Domain str u cture of phosphofructokinase 2 Figure 16.29

Conformational c hang es in cit rat e synt ha se on binding

oxa l oacetate Figure 17.10

Binding of citrate to th e iron - sulfur comp l ex of aconitase

Figure 17.12

S u cciny l CoA synthetase Figure 17.1 4

Q-cytochrome c oxidoreductase (cy t ochrome be,) Figure 18.11

Cy t och r ome c oxidase Figure 18.13

Co n se rvation of the three-dimensional structure of cytochrome c

Figure 1 8.21

ATP synthase Figure 18.25

ATP - ADP tran s l ocase Fig ure 18.38

B acte ri a l photosynthetic rea ction ce nt er Figure 19.9

Ph otosystem II Figure 19.13

Ph otosystem J Figure 19.19

Ferredoxin Figure 19.21

Ferredoxin-NADl'+ reductase Figure 19.22

A light -harvesti n g complex Figure 19.30

Rubisco Figure 20.3

Thioredoxin Figure 20.15

Glycogen phosphorylase Figure 21.6

Phosphorylase a and phosphorylase b Figure 21.9

Active site of methyl malonyl CoA mutase Figure 22.17

Ubiquitin Figure 23.2

Tetraubiquitin Figure 23.4

20S proteasome Figure 23.5

Proteasome evolution Figure 23.8

Bi osynthesis of thiamine Figure 23.9

Aspartate aminotransfe ras e Figure 23.12

H omo lo gous enzymes Figure 23.19

Fe protein Figure 24.2

MoFe protein Figure 24.3

A DNA methylase bound to a target Figure 24.12

Tryptophan synthetase Figure 24.16

3 -Ph osphoglycerate dehydrogenase Figure 24.18

Regulatory doma in formed by two subunits of 3-phosphoglycerate

dehydrogenase Figure 24.20

G lut at hi one peroxidase Figure 24.26

Carbamoyl phosphate synthetase Figure 25.3

C hann el in carbamoyl phosphate synthetase Figure 25.4

Ribonucleotide reductase R2 subunit Figure 25.10

Structure of propeller domain Figure 26.19

B - ferm and A - form DNA Figure 28.3

Z - DNA Figure 28.8

Topoisomerase T Figure 28.11

Topoisomerase II Figure 28.13

DNA polymerase Figure 28.15

Conformational change in DNA polymerase o n bind in g of a

dNTP Figure 28 19

H elicase Figure 28.23

Conserved residues among helicases Figure 28 25

A sliding DNA clamp Figure 28.26

DNA-repair enzyme AlkA Figure 28.43

ere recombinase and topoisomerase I Figure 28.50

RNA polymerase Figure 29.1

RNA - DNA hybrid separation by a str u cture within RNA

Helix stack i ng in tRNA Figure 30.5

Active site of threonyl-tRNA synt h etase Figure 30.7

Editing site in threonyl-tRNA synthetase Figure 30.8

Threonyl-tRNA synthe ta se complex Figure 30.10

Classes of am i noacy l -tRNA sy ntheta ses Figure 30.12

The ribosome at high resolution Figure 30.13

Ribosomal RNA folding pattern Figure 30.14

Transfer RNA binding sites Fig ure 30.18 Elongation factor Tu Figure 30.23

lac repressor - DNA interactions Figure 31.2

H e lix -turn-helix motif Figure 31.3

DNA recognition through 13 strands Figure 31.4

H omeodomain structure Figure 31.5

na s i e -l euc in e zipper Figure 31.6

Zinc-finger domains Figure 31.7

The lac repressor Figure 31 11

A dimer of CAP bound to DNA Figure 31.16

Nucleosome core particle Figure 31.20

H omologous his tones Figure 31.21

GAL4 binding sites Figure 31.23

Two nuclear hormone receptor domains Figure 31.26

T iga n d binding to nuclear hormone receptor Figure 31.27

Estrogen receptor - tamoxifen complex Figure 31.29

Hi stone acetyl transferase F i gure 31.30

A bromodomain Figure 31.3 1

Ferrit i n F igure 31.36

Aconitase Figure 31.39

Ankyrin repeat Figure 32.35

PAMP-recognition unit of the Toll-like receptor Figure 33.3

Immunoglobulin G Figure 33.5

Immunoglobulin fold Figure 33.12 Variable domains of the Land H chains F i gure 33.13 Complex between an F ' " fragment of an antibody and its larget ,

phosphorylcholine Figure 33.14

Antibodies against lysozyme Figure 33.15

Antibody-lysozyme interactions Figure 33.16

C la ss I M He protein Figure 33.26

C la ss 1 MHC peptide - binding s i te Figure 33.27

T-eell receptor Figure 33.29

T-eell recep l or - Class I MHC complex Figure 33.30

The coreceptor CD8 Figure 33.31

C l ass II MHC protein Figure 33.36 Coreceptor CD4 Figure 33.37

Polymorphism in class I MHC proteins Figure 33.40

HIV receptor Figure 33.42

Myosin str u cture at high resolution Figure 34.4

Myosin light chains Figure 3 4 5

"N f yosin two - stranded coiled coil Figure 34.6

H ead domain ofkinesin at high resolution Figure 34.7 Dynein h ead - domain model Figure 34.8

Lever-arm motion Figure 34.9

Neck l inker Figure 34.11 Actin Figure 34.15

Actin and hexokinase Figure 34.16

Tubulin Figure 34.22

FlageLlin Figure 34 26

Flagellar motor components Figure 34 28

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Acknowledgments

Thanks go first and for e m os t to our s tudents Not a word wa s written or an illu s tration

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detect vagueness and ambiguity We also thank our co ll eag u es who supported, advised,

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o ur co ll e ague s thro u ghout t h e world who patientl y answered o ur questi o ns and shared

their in s i g ht s into r ece nt developm e nts W e thank S u sa n J Baserga and Erica A

C hampi on of the Yal e Univers it y Sc h oo l of Medicin e for th e ir outstandi n g co

ntribu-tion s in the revi s ion of C hapter 2\) Al a n Mellor s of th e Univer s ity of G u e lph ,

Eme ritu s, d ese r ves our thanks for r ea din g eve ry chapt e r of page proof t o c h ec k for

accuracy W e also espec iall y thank th ose who s erved a s reviewer s for thi s n ew ed ition

Th e ir th o u g htful co mm e n ts, s ugg es ti o n s, a nd e n co ura ge ment h ave be e n of imm e ns e

h e lp to u s in maintainin g the excellence o f the preceding e diti o n s The se r ev i ewe r s are :

Zac h aria h Dhanarajan

J ose ph Eic h berg

K C hri sto ph e r Ga r cia

Univer s ity of Mi c h igan

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U niv e r s ity of Mi ssou ri

O li ver E Owen

Retired clinical investigator,

administra tor , and a c ademician

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x V I

Stony l3r ook U niv e r si t y U ni versi t y oJ Michigan

Takita Felder S uml e r Br e nt M Z n osko

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Workin g w ith our co ll eagues atW H Fr ee man and Co mpan y ha s b ee n a wonderful

ex-p e ri e n ce We wo uld es pecially like to acknowled ge the efforts of the f o ll ow ing people Our

d e velopmental editor, S usan Moran, h as co ntribut e d imm e n se l y to the s u ccess of this

proj ec t O ur project e ditor, Geo r g ia Le e H a dl e r , managed the fl ow of the proj ec t from final manu scr ipt to final produ c t with admirable eff i c i e n cy Th e ca r ef ul manu sc ri pt e di -

t or, Patricia Z immerman, en han ced the t e xt' s lit e rar y co n sis t e n cy and clarity Design manager Diana Blum e produ ce d a design and l ayo ut that are o r ga nizati o nall y clear and es-

theti ca ll y pleasing Ou r photo e ditor , Bian c a Moscatelli , tena c iou s l y tra c ked down n ew inl

-a ges nill Page, the illustration coo rdinator , ably ove r sa w th e r e nd e ring of new illu s trations, and S u sa n W e in , th e production manag e r , a s tut e l y handled all th e diffi c ulti es of s chedu l -

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O ur acquisitions editor, Kate Abr , was a n outstanding dir ec tor of th e pr o j ect Her

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tired , frustrated, and di scou r aged Marketi n g mavens J o hn Brit c h and Sarah Martin

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Finally , the proj ec t wou ld not have been po s sib l e without the unfailing s upport of o ur familie s specially our w ive s, Wendi e B e rg and Alison U n ger Th e ir patien ce, e n co ura ge-

m e nt , a nd e nthusiasm hav e mad e this e ndeavor po ss ible W e also thank o ur c hildr e n , A l ex,

Corey, and Monica B erg and Janina and Nichola s Tymoczk o , for their forb eara n ce and good humor and for constantly providing us a p e r s pe ct iv e on what is tr ul y imp o rtant in lif e

Ma r y K ay O r g ill

U n iv e r s it y of Mi ss ouri

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Re t ire d clini c a l i n ve s t iga t or,

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S t o n y B roo k U ni v e rsi t y U niver s i t y oj Mi c h ig an

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x-p e ri e n ce W e wo uld es p ec i a ll y like to a c knowled ge th e e ffor ts of th e f o ll o win g p eo pl e O ur

d eve l o pm e ntal e ditor , S u sa n Moran , ha s co ntribut e d imm e n s el y t o th e s u ccess o f t hi s

pr o j ec t O ur proj ec t editor , Geo rgia L ee Hadl e r , man age d th e fl ow o f th e p ro j ec t from fin a l m a nu sc ript to final produ c t with a dmirabl e ef fi c i e n cy T h e ca reful m a nu sc ript e di-

t o r , P a tri c i a Z imm e rman , e nhan ce d th e t e xt' s li tera r y co n s i s t e n c y and cl a ri ty D es i g n

m anage r Dian a Blume produ ce d a d e s ign and l ayo u t th a t ar e o r ga ni za ti o n a ll y cle ar and es

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-ag e s Bill P age , th e illu s tration c oordina to r , a bl y over s aw th e r e nd e rin g of n e w illu s trati o n s ,

a nd S u sa n W e in , th e produ c tion manag e r, a s tu tely handl e d all th e diffi c ulti es o f sc h e dul

-in g, co mpo s iti o n , a nd manufacturing M e di a edit o r A l ys i a B a k e r a nd a ss i s tant e dit o r s N i c k

T y m ocz k o and D ee na Goldman w e r e in v aluab le in th ei r mana ge m e nt of th e m e dia and

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tir e d, fru s tr ate d , a nd d i s c o ur age d M a rk e tin g m av e n s J o hn Brit c h a nd Sa rah Martin

ove r sa w th e int ro du c ti o n of thi s e diti o n t o th e a c a d e mi c w o rld W e al so th a nk th e sa l es peo pl e at W H F re e m a n and Co m pa n y for th e ir e x ce llent s u gges tion s and v i e w of th e

mark e t W e th a n k Eli za b e th W iddicom b e , Pr es id e nt o f W H f r ee man and C ompan y,

fo r neve r l os in g faith in u s

F inall y, th e proj e ct wo uld n o t h ave b ee n po ss ibl e with o ut th e unf a ilin g s upp o r t o f o ur

f a mi li es es p ec iall y o ur w i ves , Wendi e B e r g and Ali so n U n ge r Th e ir p a ti e n ce, e n c oura

ge-m e n t , and e nthu s ia s m ha ve made this e nd e avor p oss ibl e W e al so thank our c hildr e n, Al e x ,

Co r ey, and Mo ni ca Ber g and Janin a and N i c hola s T y m oc zk o, f o r th e ir f o r bear an ce and

good hum o r and f o r c on sta ntl y pro v idin g u s a per s p ec ti ve o n w hat i s trul y im po rtant in lif e

Brief Contents

Part I THE MOLECULAR DESIGN OF LIFE

1 Biochemistry: An Evolving Science 1

2 Protein Composition and Structure 25

3 Exploring Proteins and Proteomes 65

4 DNA, RNA, and the Flow of Genetic

Information 107

5 Exploring Genes and Genomes 134

6 Exploring Evolution and Bioinformatics 164

7 Hemoglobin : Portrait of a Protein in Action 183

8 Enzymes: Basic Concepts and Kinetics 205

9 Catalytic Strategies 241

10 Regulatory Strategies 275

11 Carbohydrates 303

12 Lipids and Cell Membranes 326

13 Membrane Channels and Pumps 351

14 Signal-Transduction Pathways 381

Part II TRANSDUCING AND STORING ENERGY

15 Metabolism : Basic Concepts and Design 409

16 Glycolysis and Gluconeogenesis 433

17 The Citric Acid Cycle 475

18 Oxidative Phosphorylation 502

19 The Light Reactions of Photosynthesis 541

20 The Calvin Cycle and the Pentose Phosphate

Pathway 565

21 Glycogen Metabolism 592

22 Fatty Acid Metabolism 617

23 Protein Tumover and Amino Acid Catabolism 649

Part III SYNTHESIZING THE MOLECULES OF LIFE

24 The Biosynthesis of Amino Acids 679

25 Nucleotide Biosynthesis 709

26 The Biosynthesis of Membrane Lipids

and Steroids 732

27 The Integration of Metabolism 760

28 DNA Replication , Repair , and Recombination 783

29 RNA Synthesis and Processing 821

30 Protein Synthesis 857

31 The Control of Gene Expression 892

Part IV RESPONDING TO ENVIRONMENTAL

Part I THE MOLECULAR DESIGN OF LIFE

Chapter 1 Biochemistry: An Evolving Science 1

1.1 Biochemical Unity Underlies Biological Diversity 1

Form and Function

DNA Is Constructed from Four Building Blocks

Two Single Strands of DNA Combine to form a

Covalent and Noncovalent Bonds Are Important for the

Acid Base Reactions Are Central in Many

The Sequencing of the Human Genome r sa

Genome Sequences Encode Proteins and Patterns

• • •

x v " I Contents

Polypeptide Chains Are Flexible Yet Conformationally

2.3 Secondary Structure : Polypeptide Chains Can

Fold into Regular Structures Such As the Alpha

Helix , the Beta Sheet , and Turns and Loops 40

The Alpha Helix Is a Coiled Structure Stabilized by

Beta Sheets Are Stabilized by Hydrogen Bonding

Polypeptide Chains Can Change Direction by Making

Fibrous Proteins Provide Structural Support for Cells

2.4 Tertiary Structure : Water-Soluble Proteins Fold

i nto Compact Structures with Nonpolar Cores 46

2.5 Quaternary Structure : Polypeptide Chains

Can Assemble into Multisubunit Structures 49

2.6 The Amino Acid Sequence of a Protein

Determines Its Three - Dimensional Structure 50

Amino Acids Have Different Propensities for forming

Alpha Helices, Beta Sheets, and Beta Turns 52

Protein Misfolding and Aggregation Are Associated with

Some Neurological Diseases 5.1

Protein Folding I s a Highly Cooperative Process 55

Proteins Fold by Progressive Stabilization

of lntermediates Rather Than by Random Search

Prediction of Three -Dimen s ional S tru cture from

Seqllel1ce Remail1s a Great C hallenge

Protein Modification and Cleavage Confer New

Capabilities

S5

57

APPEND1X : Visualizing Molecular Structures II: Proteins 61

Proteomes

The Proteome I s the Functional Representation

of the Genome

3.1 The Purification of Proteins Is an Essential

First Step in Understanding Their Function

The Assay: How Do We Recognize the Protein

Size, Charge, and Binding Activity 68

Proteins Can De Separated by Gel Electrophoresis

3.2 Amino Acid Sequences Can Be Determined

Proteins Can Be Specifically Cleaved into Small Pep tides

Antibodies to Specific Proteins Can Be Generated 84 Monoclonal Antibodies with Virtually Any Desired

Specificity em Be Readily Prepared 85 Proteins Can Be Detected and Quantitated by Using an

Enzyme· Linked [mmunosorbent Assay R7

Western Blotting Permits the Detection of Proteins Separated by Gel Electrophoresis 88

Fluorescent Markers Make Possible the Visualization

3.4 Peptides Can Be Synthesized by Automated

Individual Protein Components of Large Protein Complexes

Can FIe rdentified by MALDI · TOF Mass Spectrometry 94

3.6 Three-Dimensional Protein Structure Can Be Determined by X-ray Crystallography and NMR

X -ray Crystallography Reveals Three· Dimensional

N uelear Magnetic Resonance Spectroscopy Can Reveal the titructures of Proteins in Solution

Nucleotides Are the Monomeric Units

4.2 A Pair of Nucleic Acid Chains with omplementary

Sequences Can Form a Double-Helical Structure 111

The Double Helix Is Stabilized by Hydrogen Bonds and

Hydrophobic Interactions 111

The [)ouble Helix Facilitates the Accurate Transmission

Single-Stranded Nucleic Acids Can Adopt

4.3 DNA Is Replicated by Polymerases That

[)NA Polymerase Catalyzes Phosphodiester-Rond

4.4 Gene Expression Is the Transformation

of DNA Information into Functional Molecules 119

Several Kinds of RNA Play Key Roles in Gene

All Cellular RNA Is Synthesized by RNA Polymerases 120

RNA Polymerases Take Instructiuns from DNA Templates 121

Transcription Begins Near Promoter Sites and Ends at

Transfer RNA Is the Adaptor Molecules in Protein

4.5 Amino Acids Are Encoded by Groups

of Three Bases Starting from a Fixed Point 124

Messenger RNA Contains Start and Stop Signals

4.6 Most Eukaryotic Genes Are Mosaics

Chapter 5 Exploring Genes and Genomes 134

5.1 The Exploration of Genes Relies on Key Tools 135

Restriction Enzymes Split DNA into Specifi c Fragments 135

Restriction Fragments Can Be Separated by

Gel Electrophoresis and Visualized 136

DNA Can Be Sequenced by Controlled Termination

PCR Is a Powerful Technique in Medical Diagnostics,

Forensics, and Studies of Molecular Evolution

5.2 Recombinant DNA Technology Has Revolutionized All Aspects of Biology

Restriction Enzymes and DNA Ligase Are Key Tools

14 1

142

in Forming Recombinant DNA Molecules 142

Plasmids and Lambda Phage Are Choice Vectors

Bacterial and Yeast Artificial Chromosumes 145 Specific Genes Can lie Cloned from Digests of

Proteins with New Functions Can Be Created Through

5.3 Complete Genomes Have Been Sequenced

'1 ' he Genomes of Organisms Ranging from Bacteria

to Multicellular Eukaryotes Have Been Sequenced 149

New Genes Inserted into Eukaryotic Cells Can Be

Efficiently Expressed

Transgenic Animals H arbor and Express Genes That Were Introduced into Their Germ Lines

Gene Disruption Provides Clues to Cene Function

RNA Interference Provides an Additional 1 00 1 for Disrupting Gene Expression

Tumor-Ind ucing Plasm ids Can Be Used to Introduce

New Genes into Plant Cells

Human Gene Therapy Holds Great Promise

6.2 Statistical Analysis of Sequence Alignments

The Statistical Significance of Alignments Can Be

Distant Evolutionary Relationships Can Be Detected

Databases Can Be Searched to Identify

x x Contents

6.3 Examination of Three-Dimensional Structure

Enhances Our Understanding of Evolutionary

Tertiary Structure 1 s More Conserved Than

Knowledge of Three- Dimensional Structures Can

Aiu in the Evaluation of Sequence Alignments 174

Repeated Motifs Can Be Detected by Aligning

Convergent Evol ution Jllustrates Common

Solutions to Biochemical Challenges 17"

Comparison of RNA Sequences Can Be a Source

of Insight into RNA Secondary Structures 176

6.4 Evolutionary Trees Can Be Constructed

on the Basis of Sequence Information 177

6.5 Modern Techniques Make the Experimental

Ancient DNA Can Sometimes [Je Amplified and

Molecular Evolution Can Be Examined Experimentally 178

Chapter 7 Hemoglobin : Portrait of

7.1 Myoglobin and Hemoglobin Bind Oxygen

The Structure of M yoglobin Prevents the Release

of Reactive Oxygen Species 185

Human Hemoglobin Is an Assembly of Four

M yoglobin-like Subunits 186

7.2 Hemoglobin Binds Oxygen Cooperatively 187

Oxygen Binding Markedly C hanges the Quaternary

Structure of Hemoglobin 188

Hemoglobin Cooperativity Can Be Potentially

Explained by Several Models 189

Structural Changes at the Heme Croups Are

Transm itted to the Uti3 t-<>Al2 Interface 190

2,3-Bisphosphoglycerate in Red Cells Is Crucial in

Determining the Oxygen Affinity of H emoglobin 190

7.3 Hydrogen Ions and Carbon Dioxide Promote

t he Release of Oxygen : The Bohr Effect 192

7 4 Mutations in Genes Encoding Hemoglobin

Sickle-Cell Anemia Results from the Aggregation

of Mutated Deoxyhemoglobin Molecules 195 Thalassemi" Is Caused by an Imbalanced Production

The Accumulation of Free Alpha-Hemoglobin

Additional Globins Are Encoded in the [I uman Genome 197

APPENOlX: Binding M odels Can Be Formulated in

Q uantitative Terms: The Hill Plot and the

Many Enzymes Require Cofactors fur Activity 207

Enzymes May Transform Energy from One Form

8.2 Free Energy Is a Useful Thermodynamic

The Free-Energy C hange Provides Jn formation About

the Spontaneity but Not the Rate of a Reaction 208

T he Standard Free-Energy Change of a Reaction

Js Related to the Equilibrium Constant 208

Enzymes Alter O nly the Reaction Rate and Not

the Reaction Equilibrium 210

8.1 Enzymes Accelerate Reactions by Facilitating the Formation of the Transition State 211

The Formation of an Enzyme- Substrate Complex Js

the First Step in Enzymatic C atalysis 213

T he Active Sites of Enzymes Have Some Common

The Bind ing Energy Between Enzyme and Substrate

8.4 The Michaelis-Menten Equation Describes the Kinetic Properties of Many Enzymes 216

Kinetics Is the Study of Reaction Rates 216

The Steauy -State Assumption Facilit"tes a

Description of Enzyme Kinetics 217

KM and Vmax Values Can Be D etermined by

Several Means

KM and Vm Values Are Important Enzyme

Characteristics

k ",I K M Is a Measure of Catalytic Efficiency

Most Biochemical Reactions Include Multiple

-8.5 Enzymes Can Be Inhibited by Specific

Molecules

Reversible Inhibitors Are Kinetically Dist;nguishable

Irreversible Inhibitors Can Be Used to Map the

Active Site

Transition-State Analogs Are Potent Inhibitors

of Enzymes

Catalytic Antibodies Demonstrate the Importance

of Selective Binding of the Transition State

to Enzymat ic Activity

Penicillin Irreversibly Inactivates a Key Enzyme

in Bacterial Cell -\\1al l Synthesis

APPENDIX: Enzymes Are C lassified on the Basis

of the Types of Reactions That They Catalyze

Chapter 9 CatalytiC Strategies

A few Basic Catalytic Principles Are Used by

Chymotrypsin Possess a Highly Reacti ve Serine Residue 243

Chymotrypsin Action Proceed in Two Steps Linked

by a Covalently Bound Intermediate 244

Serine Is Part of a Catalytic Triad That Also Includes

Catalytic Triads Are Found in Other Hydrolytic

The Catal yt ic Triad Has Been Dissected by Sit

Cysteine Aspartyl and Metalloproteases Are O ther

Major Classes of Peptide-Cleaving Enzymes 251

Protease Inhibitors Are Important Drugs 253

9.2 Carbonic Anhydrases Make a Fast Reaction

Carbonic Anhydrase Contains a Bound Zinc Ion

Essential for Catalyti c Activity 255

Catalysis Entails Zinc Activation of a Water Molecule 256

A Proton Shuttle facilitates Rapid Regeneration

Convergent Evolution Has Generated Zinc-Based

Active Sites in Different Carbonic Anhydrases 258

9.3 Restriction Enzymes Perform Highly Specific

Cleavage Is by fn-Line Displacement of 3' -Oxygen

from Phosphorus by IVlagnesium-Activated Water 260

Restriction Enzymes Require Magnesium for

The Complete Catalytic Aparatus Is Assembled

O nly Within Complexes of Cognate DNA M olecules

Co n te nt s x X i

Type H Restriction Enzymes H ave a Catalytic Core

in Common and Are Probably Related by Horizontal

Triphosphates Are the True Substrates for Essentially

A ll t-:TP -Dependent Enzymes 26 8

P -Loop NTPase Domains Are Present in a Range of

Chapter 10 Regulatory Strategies 275

10.1 Aspartate Transcarbamoylase Is Allosterically

Allosterically Regul ated Enzymes Do Not follow

10.3 Covalent Modification Is a Means of

Phosphorylation Is a Highly Effective !V!eans of Regulating the Activities of Target Proteins 2H4 Cyclic AMP Activates Protein Kinase A by Altering the

to the Formation of a Substrate-Binding Site 290 The Generation of Trypsin from Trypsinogen Leads

to the Activation of Other Zymogens 291 Some Proteolytic Enzymes Have Specific Inhibitors 291 Blood Clotting Is Accomplished by a Cascade

Fibrinogen Is Converted by Thrombin into a Fibrin Clot 293

• •

x X II Contents

Prothrombin h Readied for Activation by

a Vitamin K-D ependenl Modification

Hemophilia Revealed an F.arly Step in Clotting

The Clolling Process Must Be Regulated

Pentoses and Hexoses Cyclize to Form Furanose

Pyranose and Furanose Rings Can Assume Different

Monosaccharides Are Joined to Alcohols and Amines

Phosphoryl ated Sugars Are Key Intermediates in

Energy Generation and Biosyntheses 310

11.2 Complex Carbohydrates Are Formed by

Sucrose Lactose and Maitose Are the Common

Glycogen and Starch Are Mobilizable Stores of Glucose 31 1

Cellulose the Major Structural Polymer of Plants

Consists of Linear Chains of Glucose Units 312

Glycosaminoglycans Are Anionic Polysaccharide

Chains Made of Repeating Disaccharide Units 312

Specific Enzymes Are Responsible for Oligosaccharide

11 3 Carbohydrates Can Be Attached to Proteins

Carbohydrates Can Be Linked to Proteins Through

Asparagine (N-Linked) or Through Serine or

Threonine to -Linked) Residues

Protein G lycosylation Takes Place in the Lumen of the

Endoplasmic Reticulum and in the Golgi Complex

Errors in Gl ycosylation Can Result in Pathological

Co nditions

Oligosaccharides Can Be "Sequenced"

11.4 Lectins Are Specific Carbohydrate-Binding

Lectins Promote [nteraction Between Cells 320

Innuenza Virus Binds to Sialic Acid Residues 32 1

Many Common Features Underlie the Diversity

12.1 Fatty Acids Are Key Constituents of Lipids 327

Fatty Acid Names Are Based on Their Parent

A Membrane Lipid Is an Amphipathic Molecule

Containing a H ydrophilic and a Hydrophobic Moiety 332

12.3 Phospholipids and Glycolipids Readily Form

Lipid Vesicles Can Be Formed from Phospholipids 334 Lipid Bilayers Are Highly Impermeable to fons

12 4 Proteins Carry Out Most Membrane

Some Proteins Associate with Membranes Through

Covalently Attached Hydrophobic Croups 340

Transmembrane Helices Can De Accuratel y Predicted

12.5 Lipids and Many Membrane Proteins Diffuse

The Fluid Mosaic Model Allows Lateral Movement but Not J{otation Through the Membrane

Membrane Fluidity Is Controlled by Fatty Acid Composition and C holesterol Content

All Hiological Membranes Are Asymmetric

12.6 Eukaryotic Cells Contain Compartments Bounded by Internal Membranes

Chapter 13 Membrane Channels and Pumps

The Expression of Transporters Largely Defines the

Metabolic Activities of a Gi ve n Cell Type

13 1 The Transport of Molecules Across

a Membrane May Be Active or Passive

M any Molecules Require Protein Transporters to Cross Membranes

Free Energy Stored in Concentration Gradients

13.2 Two Families of Membrane Proteins Use ATP

Hydrolysis to Pump Ions and Molecules Across

Digita li s Specifically Inhibit s the Na I K + Pump by

Blocking It s Depho s phorylation 35 7

Mu ltidru g Resistan ce Highlight s a Family of Memhrane

13.3 Lactose Permease Is an Archetype of Secondary

Transporters That Use One Concentration Gradient

13.4 Specific Channels Can Rapidly Transport

Patch - Clamp Conducta n ce Mea s urement s Reveal the

Act i v iti es of Single Channels 363

The St ructure of a P o tassium I on Chann e l I s an

T 'h e St ru c ture of th e Potassium I on C hann e l Reveals

the Ba s is o f I on Specificity 365

Th e Structure of th e Pota ssi um I on C hannel Explains

Voltage Gatin g Require s Substa nt ia l Conforma ti o na l

Action Potentials In tegrate the Activities of Severa l Ion

13.5 Gap Junctions Allow Ions and,small

Molecules to Flow Between

13.6 Specific Channels Increase the Permeability

• • •

Chapter 14 Signal - Transduction Pathways 381

Signal- Tran sd uction Depend s on Molecular Circ uit s 382

Activated G Proteins Tra n s mit Sig nal s by B i ndin g

G Prot eins Spontaneous l y Reset Themsel yes Throu g h

Calcium Ion Often Activates the Re g ulatory Protein

Are Central to Many Signal-Transduction

In sulin B i nding Re s ult s in the Cross-Phosphory lati on

The A ct ivat ed Insulin Receptor Kina se Init i ate s a

In s ulin Signaling I s T e rminated by the A c tion

14 3 EGF Signaling : Signal-Transduction Pathways

EGF Binding ResulLs i n the Dimerization of the

EGF Signaling Leads to the Activation of Ra s , a S mall

Activated Ras Init i at es a Protein Kina s e C a sca de 398

EGF Signa lin g Is T e rminated by Protein Pho s phata ses

14.4 Many Elements Recur with Variation in

14 5 Defects in Signal-Transduct i on Pathways

Protein Kinase Inhibit ors Can Be Ef f ect iv e Anti c an ce r