DONALD VOET
`University of Pennsylvania
`
`JUDITH G. VOET
`Swarthmore College
`
`BIOCHEMISTRY
`
`SECOND EDITION
`
`JOHN WILEY & SONS, INC.
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`I09
`
`Page 2 of 8
`
`

`
`Introduction to
`Enzymes
`
`1. Historical Perspective
`2. Substrate Specificity
`A. Stcreospecificity
`B. Geometric Specificity
`3. Cocnzymes
`
`4. Regulation of Enzymatic Activity
`5. A Primer of Enzyme Nomenclature
`
`The enormous variety of biochemical reactions that com-
`prise life are nearly all mediated by a series of remarkable
`biological catalysts known as enzymes. Although enzymes
`are subject to the same laws of nature that govern the be-
`havior ofother substances, they difl'er from ordinary chem-
`ical catalysts in several important respects:
`
`1. Higher reaction rates: The rates of enzymatically cata-
`lyzed reactions are typically factors of 10‘ to IO" greater
`than those of the corresponding uncatalyzed reactions
`and are at least several orders ofmagnitudc greater than
`those of the corresponding chemically catalyzed reac-
`trons.
`
`2. Milder reaction conditions: Enzymatically catalyzed re-
`actions occur under relatively mild conditions: temper-
`atures below l00°C, atmospheric pressure, and nearly
`neutral pH's. ln contrast, eflicient chemical catalysis
`often requires elevated temperatures and pressures as
`well as extremes of pH.
`3. Greater reaction specificity: Enzymes have a vastly
`greater degree ofspecificity with respect to the identities
`of both their substrates (reactants) and their products
`than do chemical catalysts; that is, enzymatic reactions
`
`.
`rarely have side products. For example. in the cnzy .;
`synthesis of proteins on ribosomes (Section 30-3), po ‘
`peptides consisting of well over I000 amino acid 7
`dues are made all but error free. Yet, in the che I»;
`synthesis ofpolypeptides, nde reactions and incom -.
`reactions presently limit the lengths of polypeptides ,
`can be accurately produced in reasonable yields to~l
`residues (Section 643).
`
`'
`
`-
`-
`Capacity for regulation: The catalytic activities of -
`enzymes vary in response to the concentrations of su
`stances other than their substrates. The mechanisms
`these regulatory processes include allosteric control. ‘_
`valcnt modification of enzymes, and variation of x
`amounts of enzymes synthesized.
`
`_‘
`
`-
`
`Consideration of these remarkable catalytic pr0P°T"‘5.
`enzymes leads to one ofthe central questions ofbi _
`‘I .
`try: How do enzymes work? We address this issue in '
`part of the text.
`In this chapter, following a historical review, WC "'
`mence our study ofenzyms with a discussion ofM0‘ "
`instances of enzyme action: one that illustrates 110"’ '3
`zyme specificity is manifested, and a second that 0“ ‘
`hes the regulation of enzyme activity. These ill’? b’.
`means exhaustive treatments but are intended I0 _l"3hl
`these all-important aspects of enzyme mechanlifl"
`shall encounter numerous other examples of IN“ ,«
`nomena in our study of metabolism (Chapt€fS_ '5,’
`These two expositions are interspersed with a confldegp l
`ofthc role ofenzymatic cofactors. The chapter end’ 13 a
`short synopsis ofenzyme nomenclature. In ChIlP"' S
`.
`take up the formalism ofcnzyme kinetics because W‘ V»
`of the rates of enzymatically catalyzed reactions Pm”
`indispensable mechanistic information. Finally» Ch
`POWE ‘
`
`,
`
`Page 3 of 8
`
`

`
`4 is a general discussion ofthe catalytic mechanisms em-
`loycd by enzymes, followed by an examination of the
`h;|l'llSl'l‘l5 ofseveral specific enzymes.
`P
`met
`
`1' HISTORICAL PERSPECTIVE
`
`The early history of enzymology, the study ofcnzyrnes, is
`largely that ofbiochemistry itself; ‘these disciplines evolved
`gogether from nineteenth century investigations offennen-
`“(ion and digestion. Research on fermentation is widely
`considered to have begun in l8l0 with Joseph Gay-Lus-
`,3¢'s determination that ethanol and C0, are the principal
`ucls of sugar decomposition by yeast. in 1835, Jacob
`Bflzglius, in the lirst general theory of chemical catalysis,
`poimed out that an extract ofmalt known as diastase (now
`known to contain the enzyme er-amylase; Section 10-2D)
`analyzes the hydrolysis ofstarch more etliciently than does
`sulfuric acid. Yet, despite the ability of mineral acids to
`mimic the elfect ofdiastase. it was the inability to reproduce
`most other biochemical reactions in the laboratory that led
`Louis Pasteur, in the mid-nineteenth century, to propose
`mat the processes of fennentation could only occur in liv-
`ing cells Thus, as was common in his era, Pasteur assumed
`that living systems were endowed with a “vital force" that
`permitted them to evade the laws ofnature governing inan-
`imate mattcr. Others, however. notably Justus Liebig, ar-
`gued that biological processes are caused by the action of
`chemical substances that were then known as “l'ern1ents.”
`Indeed. the name "enzyme" (Greek: en. in + zymc. yeast)
`
`Section 1.’-2. Substrate Specificity 333
`
`Although the subject of enzyinology has a long history.
`most ofour understanding of the nature and functions of
`enzymes is a product ofthe latter halfofthe twentieth cen-
`tury. Only with the advent ofmodern techniques for sepa-
`ration and analysis (Chapter 5) has the isolation and char-
`acterization ofan enzyme become less than a monumental
`task. It was not until I 963 that the iirst amino acid sequence
`of an enzyme, that of bovine pancreatic ribonuclease A
`(Section I4-IA), was reported in its entirety, and not until
`I965 that the first X-ray structure ofan enzyme, that olhen
`egg white lysozyine (Section I4-ZA), was elucidated. In the
`years since then, several thousan
`'
`lied and characterized to at least some extent and the pace
`of this endeavor is rapidly accelerating.
`
`2. SUBSTRATE SPECIFICITY
`
`The noticevalentjbrces through which mbstrates andother
`molecules bindto enzymes are identical in character (0 the
`forces that dictate the conformation: ofthe proteins them-
`selves (Section 7-4): Both involve van der Waals, electro-
`static, hydrogen bonding. and hydrophobic interactions. In
`general. a substrate—binding site consists of an indentation
`or clefl on the surface of an enzyme molecule that is com-
`plementary in shape to the substrate (geometrical comple-
`mentarity). Morcover, the amino acid residues that form
`the binding site are arranged to interact specifically with the
`substrate in an attractive manner (electronic complemen-
`tarity; Fig 12-1). Molecules that difl‘er in shape or func-
`
`the yeast itself, that catalyzes the reactions offermentation.
`Nevertheless. it was not until I897 that Eduard Buchner
`obtained a cell-free yeast extract that could carry out the
`synthesis ofelhanol from glucose (alcoholic fermentation;
`Section I6-3B).
`Emil Fischer’: discovery,
`Zltmes can distinguish betwee
`lbe fomtulation ofhis lock-and-key hypothesis: Thespeci-
`ficityofan enzyme(theIockjforitssubstrate(theIcey)arises
`from their geometrical!y complementary shapes. Yet, the
`<-jhemical composition of enzymes was not fumly estab-
`lished until well into the twentieth century. In I92 ,
`ho crystallized the liist enzyme, jack
`"59. which catalyzes the hydrolysis of urea to NH, and
`onstrated that these crystals consist of protein.
`mner's preparations were somewhat impure, how-
`
`“fa Ogical experiencesince then hasamplydemonstrated
`' °"2Ymes are proteins (although it has recently been
`Isl.:’_"’" thatsomespeciesofRNAalsohavecatalyticproper-
`» Section 29-4B).
`
`Page 4 of 8
`
`

`
`334 Chapter 12. Introduction to Enzymes
`
`tional group distribution from the substrate cannot produc-
`tively bind to the enzyme; that is,
`they cannot fonn
`enzyme-substrate complexes that lead to the formation or
`products. The substrate-binding site may, in accordance
`with the lock-and-key hypothesis, exist in the absence of
`bound substrate or it may. as suggested by the induced fit
`hypothesis (Section 9-4C), form about the substrate as it
`binds to the enzyme. X-Ray studies indicate that the sub
`strate-binding sites ofmost enzymes are largely preformed
`but that most of(Item exhibit at least some degreeofinduced
`fit upon binding substrate.
`
`A. Stereospecificity
`
`Enzymes are highly specific both in binding chiral sub-
`strates and in catalyzing their reactions. This stereospeci-
`ficity arises because enzymes, by virtue of their inherent
`chiraiity (proteins consist of only L-amino acids), form
`asymmetric active sites. For example, trypsin readily hy-
`drolyzes polypeptides composed of L-amino acids but not
`those consisting of D-amino acids. Likewise, the enzymes
`involved with glucose metabolism (Section I6-2) are spe-
`cific for o-glucose residues.
`
`Enzynros are absolutely stercospecijrc in the rcactiom
`they catalyze. This was strikingly demonstrated for the case
`of yeast alcohol dehydrogenase (YADH) by Frank West.
`heimer and Birgit Vennesland. Alcohol dehydrogenase cat-
`alyaes the interconversion ofethanol and acetaldehyde ac.
`cording to the reaction:
`
`0
`
`h +
`I
`wmrr
`+
`CH;.CH,Ol-I + NAD «ti CH,.CH + NADH + II
`Ethanol
`Acotnldohydc
`
`The structures of NAD* and NADH are presented in Fig,
`12-2. Ethanol, it will be recalled, isa prochiral molecu|e(sc¢
`Section 4-2C for a discussion of prochirality):
`
`9H
`
`Plprrr-S .1 —'H[It'r» It
`
`CH3
`
`Ethanol’s two methylene H atoms may be distinguished if
`the molecule is held in some sort ofasymmetric jig (Fig.
`12-3). The substrate-binding sites ofenzymes are, oft:0ur.rc,
`
`Oxidized lorrn
`
`Reduced form
`
`OH
`
`ll
`
`Nicotinurnidc
`
`r,
`Iii
`I
`\+..,.N
`
`M ti
`\
`
`o
`.(‘L
`
`Nil: + 11*
`
`-{fa NH,
`
`2f
`
`-1» 2In.r=‘
`
`J’
`
`\ N
`
`Aden osi no
`
`.\
`R
`
`II
`l’()
`
`Nicotinnmido adenine dinuclcotidc (NA.D"')
`Nicotitmtnidc adenine dlnucleotitlc phosphate (NADl"”)
`
`FIGURE I2-2. The structures and reactions ofnicotinamlde
`adenine dinucleotide (NA D*) and nicotinamide adenine
`dinuclcotide phosphate (NADP*). Their reduced forms are
`NADH and NADPH. [In the older literature they are termed
`tllphosphopyridine nucleotide (DPN*) and triphosphopyridine
`nucleotide ('l'PN*) and their reduced forms are symbolized
`DPNH and TPNI-L] These substances. which are collectively
`
`.
`referred to as the nicotinarnidc eoenzymes or pyridine
`nucleotides (nicotinamide is a pyridine derivative) l‘unction_. "5 '5
`indicated in later chapters, as intracellular carriers of reducIIlB_
`equivalents (electrons). Note that only the nicotinumidc l'll'|9 '5
`changed in the reaction. Reduction formally involves the
`transfer of two hydrogen atoms (H - ), although the actual
`reduction may occur via a different mechanism.
`
`Page 5 of 8
`
`

`
`Section I2-2. Stibxtratcspmjficity 335
`
`is quantitatively transferred from the NADD to the acet-
`aldehyde to fonn the product ethanol:
`
`O
`n
`CHQCH + |
`
`ll
`
`I)
`\.
`
`|
`
`’l‘n
`
`0
`II
`C‘Nn,
`'
`
`YADH
`+ 11* -.——-—-‘
`
`on
`
`1|--(1:-I) + NAB’
`
`EH3
`
`3. ifthe enantiomer of the foregoing CH,CI-IDOH is made
`as follows:
`
`0"
`0
`3
`+ man
`II
`(‘I~l,,CD + NADI-I + H ; D'-Q'-'H + NAD
`
`+
`
`c'H;.
`
`none of the deuterium is transferred from the product
`ethanol to NAD* in the reverse reaction.
`4. If, however, this ethanol is converted to its tosylate and
`then inverted by SN2 hydrolysis to yield the enantio-
`meric ethanol,
`
`p -Tolucncsulfonyl
`chloride
`(tosyl chloride)
`
`CH3
`
`I-lC‘l
`
`CH3
`
`_L
`
`4»
`
`IO
`
`+
`
`H
`
`II
`
`0 l
`
`D C‘H
`IC
`
`“J
`
`o—c—H
`
`en,
`
`the deuterium is again quantitatively transfened to
`NAD" in the YADH reaction.
`
`The foregoing observations. in addition to showing that
`there is direct hydrogen transfer in the YADH reaction
`(Experiments I and 2), indicate that the enzyme distin-
`guishes between the pro-S and pro-R hydrogens of ethanol
`as well as the st’ and re faces of the nicotinamide ring of
`NAD* (Experiments 2-4). It was later demonstrated, by
`stereospecific syntheses, that YADH transfers the pro-R
`
`12-3. The specific attachment of a prochiral center
`,9 an enzyme-binding site permits the enzyme to diflcrentiate
`. mum pt-ochiral groups.
`
`-.,
`‘
`
`,3‘
`.w
`
`just such jigs since they immobt'lt':e the reacting groups of
`the .Subslra(t.' on the enzyme surface.
`Weslhcimcr and Vennesland elucidated the stereospe-
`cihc nature of the YADH reaction through the following
`series of experiments:
`
`1. If the YADH reaction is carried out with deuterated
`ethanol. the product NADH is deuterated:
`
`0 3
`
`H
`
`\ \N"-.-
`/
`
`NAn*
`
`CH1?“ OH
`
`+
`
`0
`’l
`
`D I!
`\‘
`
`O
`ll
`(‘x
`
`cumn + [f:H/ N“:+H+
`NI
`R
`
`N
`NADD
`ch‘?'°ll|1at the nicotinamide ring of NAD* is also pro-
`tra,
`
`isolating this NADD and using it in the reverse
`‘
`r
`°“°"°n to reduce normal acetaldehyde, the deuterium
`
`Page 6 of 8
`
`

`
`336 Chapter 12. Introduction to Enzymes
`
`hydrogen of ethanol to the re face of the nicotinamide ring
`of NAD* as is drawn in the preceding diagram.
`The slereospccrficity of YADH is by no means unusual.
`As we consider biochemical reactions we shall find that
`nearly all enzymes that participate in chiral reactions are
`absolute!y riereospecrjfic.
`
`Stereospeclficity In the NADH-Dependent
`Dehydrogennses May Have Functional Significance
`In our exploration of metabolism, we shall encounter
`numerous species of NADH-dependent dehydrogenases
`that function to reduce (or oxidize) a great variety of sub-
`strates These various dehydrogenases are more or less
`equally distributed between those transferring the pro-R
`(rekside) and the pro-S (31-side) hydrogens at C4 of NADH
`(also known as A-side and B-side transfers).
`
`cates that at least some of the side chains responsible rm.
`YADI-l’s stereospecilicity are not essential for catalysis and
`hence strengthens the argument that stereospecificity in me
`dehydrogenases has functional significance.
`
`B. Geometric Specificity
`
`The stereospecificity ofenzymes is not particularly SllI'p1-is.
`ing in light ofthe complementarity ofan enzymatic binding
`site for its substrate. A substrate of the wrong chirality will
`not fit into an enzymatic binding site for much the same
`reasons that you cannot fit your right hand into your lefi
`glove. In addition to their rrereospectficiry, however, mos,
`enzymas are quiteselective about the identities ofthe chem}.
`calgroups on their substrates. Indeed, such geometric spee.
`ificity is a more stringent requirement than is stereospeci.
`ficity. After all. your left glove will more or less fit left hand;
`that have somewhat different sizes and shapes than your
`own.
`
`Yet, despite the fact that sr'- and re-side hydrogen transfers
`to or from the nicotinarnide ring yield chemically identical
`products. a particular specificity of transfer is rigidly main-
`tained within classes of dehydrogenases catalyzing similar
`reactions in diflerent organisms. indeed, dehydrogenases
`that catalyze reactions whose equilibrium constants with
`their natural substrates in the direction of reduction are
`<l0‘”M almost always uansfer the nicotinamide‘s pro-R
`hydrogen, whereas those with equilibrium constants
`>l0"°M generally transfer the pro-S hydrogen. Why has
`evolution so assiduously maintained this stereospecilicity?
`Is it simply the result ofa historical accident or does it serve
`some physiological function?
`The NADH hydrogen transferred in a given enzymatic
`reaction is almost certainly that on the side of the nicotin-
`amide ring facing the substrate. It has therefore been widely
`assumed that the stereospecificity in any given class of de-
`hydrogenases simply arose through a random choice made
`early in evolutionary history. Once made, this choice be-
`came “locked in." because flipping a nicotinamide ring
`about its glycosidic bond in NADH would result, it was
`presumed, in its carboxarnide group obstructing catalyti-
`cally essential residues on the enzyme.
`in an effort to shed light on this matter, Steven Benner
`mutated YADH in a manner that the X-ray stmcture ofthe
`closely similar enzyme horse liver alcohol dehydrogenase
`(LAD!-I) suggests permits the 3:’ face of nicotinamide to
`bind to the enzyme without interfering with catalysis. The
`resulting mutant enzyme (Leu 182 - Ala) makes one
`stereochemical “mistalre" every 850,000 turnovers vs one
`mistake every 7 billion turnovers for wild-type (unmutated)
`YADH. This 8000-fold decrease in stereospecificity indi-
`
`_‘
`
`A
`
`._
`
`A
`
`j,
`. I
`
`‘
`
`I
`
`V‘
`
`Enzymes vary considerably in their degree ofgeometric
`specificity. A few enzymes are absolutely specific for only
`one compound. Most enzymes, however, catalyze the rear.
`tions ofa small range of related compounds. For example,
`YADH catalyzes the oxidation of small primary and sec-
`ondary alcohols to their corresponding aldehydcs or ice-
`tones but none so elliciently as that ofethanoL Even melll-
`anol or isopropanol, which difl‘er from ethanol only by the
`deletion or addition ofa CH, group, are oxidized by YADH
`at rates that are, respectively, 25-fold and 2.5-fold slower
`than that for ethanol. Similarly, NADP", which difl'ers
`from NAD‘* only by the addition ofa phosphoryl group at
`,
`the 2' position of its adenosine ribose group (Fig. I2-2),
`does not bind to YADH. On the other hand, there are many _-
`enzymes that bind NADP* but not NAD*.
`Some enzymes, particularly digestive enzymes, are so
`permissive in their ranges ofacceptable substrates that tbeif
`'
`geometric specilicities are more accurately described 815
`preferences. Carboxypeptidase A, for example, catalyzf-5 '
`the hydrolysis of C-terminal peptide bonds to all residues .
`except Arg, Lys, and Pro ifthe preceding residue is not P10
`(Table 6-1). However, the rate of this enzymatic reaction
`varies with the identities ofthe residues in the vicinity ofthe
`C-terminus ofthe polypeptide (see Fig. 6-5). Some enzym°5 ‘
`are not even very specific in the type of reaction they cal?‘
`lyze. Thus chymotrypsin, in addition to its ability to 11105"
`ate peptide bond hydrolysis. also catalyzes ester bond hi‘
`drolysis.
`0
`O
`II
`*
`[1
`ll
`no mm + I-I20 if-'-'L RC—0" + u
`Peptide
`
`. 5 '
`
`0
`0
`1
`ehymotrypsin
`I
`RC Oil + H20 —f>RC- 0' + HOR
`Ester
`H+
`
`Page 7 of 8
`
`

`
`T
`
`‘M the gcyl group acceptor in the chymotrypsin re-
`,
`e'd not be water: amino acids, alcohols. or ammo-
`‘
`;,1of°°
`action
`“£350 act in this capacity. it should be realized, how-
`l1 *
`M _
`’ niflwn [Such pcrmissiveness is much more the exception
`4
`lmmic, Indeed, most intracellular enzymes function
`
`.33 CQENZYMES
`:2.
`cs catalyze a wide variety of chemical reactions.
`iv" Enzyni-“notional groups can facilely participate in acid-
`Thwmctjons, form certain types of transient covalent
`based‘ and take part in charge—charge interactions (Sec-
`bag 14.1). They are, however. less suitable for catalyzing
`“idation-reduction reactions and many types of group-
`3:“ (dons, they can onlydo so in association with small mole-
`‘I'wk mfmogs, which essentially act as the enzymes
`~' Fciiemical teeth."
`_
`:‘ cofactor: may be metal ions. such as the Zn“ required
`"’ me catalytic activity ofcarboxypeptidase A, or organic
`_fi-iolecules known as coenzyines such as the NAD* in
`impfl (Section I2-2A). Some cofactors,
`for instance
`73 AD*, are but transiently associated with a given enzyme
`. molecule so that, in effect, they function as cosubstrates.
`"other cofactors, known as prosthetic groups. are essentially
`.314 H‘ ently associated with their protein. often by cova-
`Ei t bonds. For example, the heme prosthetic group of he-
`.globin is tightly bound to its protein through extensive
`liydrophobic and hydrogen bonding interactions together
`-with a covalent bond between the heme Fe“ ion and His F8
`~ . (Sections 9-IA and 28).
`1".‘ Coenzymes are chemically changed by the enzymatic re-
`«»_actions in which they participate. Thus. in order to com-
`
`. Tiii: CDI\ll\ION Cocuzvmss
`
`Reaction Mediated
`
`Section Discussed
`
`Carboxylation
`Alkylation
`
`Acyl transfer
`Ox idation-
`reduction
`Acyl transfer
`Oxidation-
`reduction
`Amino group
`tmnsfcr
`
`One-carbon group
`transfer
`Aldehyde transfer
`
`21- IA
`23-2E
`
`I9-2A
`I-1-4
`
`I9-2A
`I2-2A
`
`24-IA
`
`24-4D
`
`I6-3B
`
`.S’t't.'ti'tm I2-3. Cotvizymes 337
`
`plete the catalytic cycle. the coenzyme must be returned to
`its original state. For prosthetic groups, this can occur only
`in a separate phase ofthe enzymatic reaction sequence. For
`transiently bound coenzymes. such as NAD*, however, the
`regeneration reaction may be catalyzed by a different en-
`zymc.
`
`A catalytically active enzyme —cofactor complex is called
`a holoenzyme. The enzymatically inactive protein resulting
`from the removal of a holoenzyn1e’s cofactor is referred to
`as an apoenzyiiie; that is,
`
`apoenzyme (iiiaciive) + cofactor
`
`—-=2 holoenzyme (aaive)
`
`Table I2-I lists the most common coenzymes together
`with the types of reactions in which they participate. We
`shall describe the structures of these substances and their
`reaction mechanisms in the appropriate sections ofthe text.
`
`Many Vitamins Are Coenzyine Precursors
`Many organisms are unable to synthesize certain por-
`tions of essential cofactors and therefore these substances
`must be present in the organisms diet: thus they are vita-
`mins. In fact, many coenzymes were discovered as growth
`factors for microorganisms or substances that cure nutri-
`tional deficiency diseases in humans and animals For ex-
`ample, the NAD* component nicotlnainitle (alternatively
`known as niaeinamide) or its carboiiylic acid analog nico-
`tinic acid (niacin; Fig. I2-4), relieve the dietary deficiency
`disease in humans known as pellagra. Pellagi-.1. which is
`characterized by diarrhea. dermatitis. and dementia, was
`endemic in the rural Southern United States in the early
`twentieth century. Most animals. including humans, can
`synthesize nicotinamide from the amino acid tryptophan
`(Section 26-6A). The corn-rich diet that was prevalent in
`the rural South. however, contained little available nicotin-
`amide or tryptophan from which to synthesize it. [Corn
`actually contains significant quantities ofnicotinamide but
`in a fonn that requires treatment with base before it can be
`intestinally absorbed. The Mexiain Indians, who are
`thought to have domesticated the corn plant. customarily
`soak corn meal in lime water—dilute Ca(OH), solution -—
`before using it to bake their staple food, tortillas]
`
`0
`
`C\Nl~l.
`
`/
`
`I
`
`N
`Nicotintimido
`lniuclnnrnidcl
`
`I \ (1\Oll
`
`/
`
`N
`Nicntinic at.-id
`tniucinl
`
`FIGURE I2-4. The structures of nicotinamide and nicotinic
`acid. These vitamins form the redox-active components of the
`nicotinamide coenzymes NAD*' and NADP‘ (compare with Fig.
`12-2).
`
`Page 8 of 8