BIOCHEMISTRY
`
`FIFTH EDITION
`
`New York
`
`Jeremy M. Berg
`Johns Hopkins University School of Medicine
`
`John L. Tymoczko
`Carleton ( -ollege
`
`Lubert Stryer
`Stanford University
`
`Web content by
`Neil D. Clarke
`{
`cT
`rt
`r
`f Medicine
`
`ne
`
`hns [Hf
`oy
`
`W. H. Freeman and Company
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`Enzymes: Basic Concepts and Kinetics
`
`HO
`
`Oy, Ca
`Aeguaiein
`
`Ho
`
`Oo
`wm
`WW
`
`NH + CO, + GO 186 om)
`
`The activity of an enryme is responsible for the glow of the
`luminescent jellyfish at lett. The ennme sequenn catalynes the
`qaidaion of a
`compound by caygen in the pevence of calcu to
`thease CO are Her (fue) Feed Bavendaen Peter Arreoie|
`
`to Coenrymes
`
`Enzymes, the catalysts of biological systems, are remarkable molecular de
`vices that determune the patterns of chemical transformations. Theyalso
`mediate the transformation of one form of energy into another. The most
`striking characteristics of enzymes are ther catalytic power
`and specificity Catalysis takes place at a particular site on
`the enzymecalled the actrie site. Nearly all known enzymes
`are protems. However proteins do not have an absolute mo
`nopoly on catalysis;
`the discovery af catalytecally active
`RNA molecules provides compelling evidence that RNA
`‘was an early biocatalyst
`(Section
`2.2.2
`Proteins as a class of macromolecules are hechly effec
`:
`tive catalysts for an enormous diversity of chemical rea
`tions because of their
`capacity te specifically bind a wery unde
`range af molecules,
`By utilizing the full repertoire
`of inter
`molecular forces, enzymes bring substrates together im an
`optimal orientation,
`the prelude to making and breaking
`chemical bonds. They
`catalyze reactions by stabilizing tran
`Htion states,
`the highest-cnergy
`species
`reaction path
`ways. By selectively stabilizing a transition state, an enzyme
`|
`determines which one of
`several potential
`chemucal rea
`Hons actually takes
`pla
`—— ,
`——
`
`> 8.1 Enzymes Are Powerful and Highly
`Specific Catalysts
`? 6.2 Free Energy ts a Useful
`The
`mac Function
`for Understanding Enrymes
`* 6.3 Enzymes Accelerate Reactions
`by Facilitating the Formation of the
`Transition State
`* 6.4 The Michaelis-Menten Model
`Accounts for the Kinetic Properties
`of Many Enrymes
`6.5 Enrymes Can Be Inhibited
`by Specific Molecules
`6.6 Vitamins Are Often Precursors
`
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`ATP + NMP == ADP + NDP
`
`8.1.3 Enzymes Are Classified on the Basis
`of the Types of Reactions That They Catalyze
`eer Ceopatnouaa ee Many enzymes have common names that provide little
`aiuiael dahabaiiba a Cit” ered
`information about
`the reactions that they catalyze. For
`example, a proteolytic enzyme secreted by the pancreas
`mec alled trypan Mira other enzymes are famed low their substrates and
`for the reactions that they catalyze, with the suffix “ase” added. Thus, an
`ATPase is an enzyme that breaks down ATP, whereas ATP synthase ia
`an enzyme that ayntheazes ATP.
`in 1964
`lo bring some consistency to the classification of enzymes,
`the International Union of Biochemistry established an Enzyme Commis-
`sion to develop a nomenclature for enzymes. Reactions were divided inta
`ax major groups numbered 1 through 6 (Table 5.3). These groups were
`subdivided and further subdivided, so that a four-digit number preceded
`by the letters
`EC
`for Enzyme Commission could precisely identify all
`enzyme
`
`as an example nucheoside monophosphate (NMP) kinase, an
`(Consider
`nme (hat we will Canine in detail in the nest chapter (Section 94)
`lt
`italyzes the following reaction
`
`
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`(192)
`CHAPTER @ + Enzymes: Basic Concepts and
`hinatet
`
`Cofactors that are small organic molecules are called coenzymes, Often
`derived from vitamins, coenzymes can be either tightly or loosely bound to
`the enzyme, Lf tightly bound, they are called prosthetic groups. Loosely as-
`sociated coenzymes are more like cosubstrates because they bind to and are
`released from the enzyme just as substrates and products are. The use of the
`same coenzyme by a variety of enzymes and their source in vitamins sets
`coenzymes apart from normal substrates, however. Enzymes that use the
`same coenzyme are usually mechanistically similar. In Chapter 9, we will
`examine the mechanistic importanceofcofactors to enzyme activity. A more
`detailed discussion of coenzyme vitamins can be found in Section 4,6,
`
`8.1.2 Enzymes May Transform Energy
`from One Form into Another
`
`In many biochemical reactions, the energy of the wactants
`wconverted with high efficiency into a different form. For ex-
`ample,
`in photosynthesis, light energy is converted into
`chemical-bond energy through an ion gradient. In mito:
`chondria, the free energy contained in amall molecules de-
`rived from food is converted first into the free energy ofan
`ion gradient and then into a different currency,the free en-
`ergy of adenosine triphosphate. Enzymes may then use the
`chemical-bond energy of ATP in many ways. The enzyme
`myosin converts the energy of ATP into the mechanical en-
`ergy of contracting muscles. Pumps in the membranes of
`cells and organelles, which can be thought of as enzymes
`that move substrates rather than chemically altering them,
`create chemical and electrical gradients by using the energy
`of ATPto transport molecules and ions (Figure 8.2), The
`molecular mechanisms of these energy-transducing en-
`#ymes are being unraveled. We will see in subsequent chap:
`ters how unidirectional cycles of discrete stepa—binding,
`chemical transformation, and release—lead to the conver-
`sion of one form of energy into another
`
`
`
`
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`TABLE 6.4 Relation between AG"
`and €,,,
`(at 75°C)
`is
`
`Aci
`kealmol”'
`
`kJ/mol
`
`The equilibrium constant under standard conditions, K’,.,, 1 defimed as
`clip
`4/6
`
`k
`
`i
`
`Substituting equation 4 into equation 3 gives
`AG
`RT Ink
`;
`
`AG
`
`1 303RT log,,,K’
`
`which can be rearranged to give
`
`K.
`
`Substituting R = 1.987 *
`sponding to 25°C) gives
`
`10
`
`deg”
`
`' and T
`
`= 298 K (corre
`
`K
`
`sa-8
`
`’
`
`in
`
`6G°" =
`
`—2.303RT log,
`
`ms = Ley x
`
`10
`
`«
`
`J98 =
`
`how (0.04075
`
`= +1.80 kcal mol
`
`“(7.53 k] mol
`
`the reaction is endergonc, DHAP will not spon
`Under these conditions,
`tancously convert to GAP
`Sow let us-calculate AG; for this reaction when. the initial concentration
`of DHAP w 2?
`=
`10
`M and the
`initial concentration of GAP ws
`3
`107° M. Substituting these values into equation | gives
`
`WART low
`
`x 10°°M
`~«
`10° M
`
`coupling of react
`
`where AG*" is here expressed in kilocalories per mole because of the choice
`of the units for R in equation 7. Thus, the standard free energy and the equi
`librium constant of a reaction are related by a simple expression. For ex-
`ample, an equilibrium constant of 10 gives a standard free-energy change
`of —1.36 keal mol
`(—5.69 kJ mol”
`') at 25°C (Table 8.4). Note that, for
`each 10-fold change in the equilibrium constant,
`the AG*’
`changes by
`1.46 keal mol~*(5.69 k] mol
`As an example,
`let us calculate AG°* and AG for the isomenzation of
`dikydroxyacetone phosphate
`(DOHAP)
`to glyceraldehyde 3-phosphate
`(GAP). This reaction takes place in glycolysis (Sectoon 16.1.4). At equilibrium
`the ratio of GAP to DHAP is 0.0475 at 25°C (298 K) and pH 7, Hence
`Keg = 0.0475, The standard free-energy change for this reaction is then
`calculated from equation |
`
`An
`
`Lo
`
`+ that
`
`the tsomerizat
`1rd
`the
`This negative value for
`qusly when
`the
`apor
`ur
`can
`fo GAP is exergonic
`and
`ncentrations. Note that 4tr for
`this re
`Present at the alorestated
`sitive.
`if
`mportant
`to
`str
`fegative, although Ati
`AG: for a reactionwlary
`i
`Centrale: of
`the
`reactant
`ha prac [he
`crote
`reaction is ACs,
`not
`hot spontancous
`|
`concentrations ot
`
`1
`
`
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`
`The equilibrium constant under standard conditions, K’,.,, 1 defined as
`
`cip
`
`AG)
`Substituting equation 4 into equation 3 gives kealmol™'=kJ/mol!
`
`4AG*
`= —RTIinK’,,
`
`4/B
`
`TABLE 6.4 Relation between iG*
`and K,,, (at 25°C)
`-—————_
`
`Coupling of reactions t
`
`the reaction is endergonic. DHAP will not spon
`Under these conditions,
`tancously convert to GAP
`Now let us calculate AG for this reaction when the initial concentration
`of DHAP is 2 * 10°° M and the
`initial concentration of GAP ww
`3
`10-7" M Substituting these values into equation | gives
`"h
`7x 10°
`
`
`~ 2 LO M
`
`AG
`
`, WART log, K'
`
`which can be rearranged to give
`
`K ‘
`
`TT
`
`4
`
`Substituting R= 1.987 *
`sponding to 25°(") gives
`
`10°*
`
`keal mol”'
`
`deg”! and T = 298 K (corre
`
`kK"
`
`17% “
`
`(fk
`
`where AGs*" is here expressed in kilocalorves per mole because af the chrwce
`ofthe wnits for R in equation 7. Thus, the standardfree energy andthe equi
`;
`I
`P
`librium Constant of a reactron are related by a simple expression. For ex-
`ample, an equilibrium constant of 10 gives a standard free-energy change
`of —1.36 keal mol
` (—3.69 k] mol‘) at 25°C (Table 8.4). Note that, for
`q
`ges
`by
`:
`each 10-fold change in the equilibrium constant, the AG*’ changes by
`1.36 keal mol™' (5.69 kJ mal
`As an example,
`let us calculate AG** and AG for the isomerization of
`dihydroxyacetone phosphate (DHAP)
`to glyceraldehyde 4-phosphate
`(GAP). This reaction takes place in glycolysis (Section 16.1.4), At equilibrium
`the ratio of GAP to DHAP is 0.0475 at 25°C (298 K) and pH 7. Hence
`cr]
`Kg = 0.0475. The standard free-energy change for this reaction is then
`calculated from equation 6
`
`4G*" =
`
`-J ART low A
`
`| OR?
`2.503 *
`= +1.480 kcal mol
`
`«
`
`x 08 &
`10
`'(7.53 kJ mol‘)
`
`long
`
`(0.0475
`
`AG=1.80 keal mol + 2.303RT log
`
`= 1.80 kcal mol
`
`49 keal mol
`
`69 bocal mod
`
`2.89 k] mol
`
`the isomerization of DHAP
`‘This negative value for the AG indicates that
`to GAPis exergor
`nd Cn GCCur spontaneously when. these SCs af
`present at the aforestated
`concen
`Note that At; for this rea
`fegative, although Ad:
`is
`AG for a faction i
`lorger
`centration: of the reacta
`reaction is AG:, not Ads
`not spontancous based o
`Concentrations of
`read
`
`l
`
`
`
`
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`196
`CHAPTER & + Enzymes: Basic Concepts and
`Kinehecs
`
`8.2.3 Enzymes Alter Only the Reaction Rate and Not the
`Reaction Equilibrium
`
`Hecause enzymes are such superb catalysts, it 15 tempting to ascribe to them
`powers that they do not have, An enzyme cannot alter the laws of thermo
`dynamics and conseyguently cannot alter the equilibrium of a chemical reac
`tion. This inability means that an enzyme accelerates the forward and re-
`verse reactions by precisely the samefactor Consider the interconversion
`of A and B. Suppose that, in the absence of enzyme, the forward rate con-
`stant (kp) is 10°* s~'
`and the reverse rate constant (ky) is 10°" s_'. The
`equilibrium constant Kis given by the ratio of these rate constants
`
`K =
`
`A,
`
`Ry
`
`nm 100
`
`The equilibrium concentration of Bis 100 times that of A, whether or not
`enzymeis present. However, it might take considerable time to approach
`this equilibrium without enzyme, whereas equilibrium would be attained
`rapidly in the presence of a suitable enzyme. Enzymes accelerate the attain-
`ment of equilibria but do not shift their positions. The equilibrium position is
`a function only of the free-energy difference betuwen reactants and products.
`
`8.3 ENZYMES ACCELERATE REACTIONS BY FACILITATING
`THE FORMATION OF THE TRANSITION STATE
`
`and vis the
`
`The free-energy difference between reactants and products accounts for the
`equilibrium of the reactoon, but enzymes accelerate how quickly this equi-
`librium is attained, How can we explain the rate enhancement in terms of
`thermodynamics? To do so, we have to consider not the end points of the
`reaction but the chemical pathway between the end points
`\ chemical reaction of substrate 5 to form product P goes through a
`transtiion state 5°
`that has a higher free energy than does either 5 or FP. The
`double dagger denotes a thermodynamic property of the transition state,
`| he
`transition state is the mast ee lcd Mk Cot cupied specws along the reaction
`pathway because it is the one with the highest free energy. The difference
`in free energy between the transition state and the substrate is called the
`Cobbs free energy of activation or simply the activation energy, symbolized
`by AG,
`as mentioned in Section §.2.1 (Figure 8.3)
`
`Transiteen state, 5
`
`FIGURE 6.5 Enrym
`activation energy
`react
`energy
`
`air
`
`Ls
`
`tay
`
`does not enter into the final 4G
`> that the energy of activation, or ACr*,
`i for the reaction, because the energy input required to reach the
`returned when the transition etate forme the product The
`barrier immediately suggests how enzymes enharice re
`yout altering AG of
`the reaction: enzymes function to lower
`ergy,
`or,
`in other words, enzwmes facilitate the formation of
`
`lerstanding how enzymes achveve this facilitateon is
`ri
`staae
`und the substrate
`(5) are in equilibrium
`
`formation of S*,
`
`
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`

`ontnbuted by
`
`1. The spectroscopic characteristics of many enzymes and substrates change
`on formation of an ES complex. These changes are particularly striking if
`the enzymecontains a colored prosthetic group. Tryptophan synthetase, a
`bacterial enzyme that contains a pyridoxal phosphate (PLP) prosthetic
`group, provides a nece illustration. This enzymecatalyzes the synthesis of
`L-tryptophan from L-serimne and incole-derivative. The addition of L-serine
`to the enzyme produces a marked increase in the fluorescence of the PLP
`group ( Figure §.6), The subsequent addition of indole, the second substrate,
`quen hes this Muorescence to a level even lower than that of the enzyme
`alone, Thus, Muorescence spectroscopy reveala the existence of an en-
`zyme-serine complex and of an enzyme-serine-indole complex. Other
`spectroscopic techniques, auch as nuclear magnetic resonance and electron
`spin resonance, also are highly informative about ES interactions
`
`}H cure 6.5 Strocture of an enryme-substrate complet. (Let) The enyme
`cytectiorme PSO i illustrated bound to its subsinate camphor
`(Right) im the active site,
`the substrate no wurounded by metidues from the ery Note alvo the presence of a
`here colactor
`
`Serine
`
`reactions. A new technique, time-resolwed crystallography, depends an co-
`erystallizing a photolabile substrate analog with the enzyme. The substrate
`analog can be converted to substrate light and images of the enzyme-
`substrate complex are obtained in a fraction of a second by scanning the
`ervetal with intense poly hromatic x-rays from a synchrotron
`
`8.3.2 The Active Sites of Enzymes Have Some Common Features
`Phe active site
`of an enzyme
`ia the region that binds the substrates (and the
`7,
`ifany).
`It also contains the residues that directly participate in the
`making and breaking of bonds. These rescues are called the catalytic groups
`e¢,
`the mteractiom of the enevew and substrate at the achiia ote pre
`formation of
`the
`transition state.
`The active site is the region of the
`hat moewt
`dir
`owers
`the AGr’
`of the reaction, which results in
`fenzyme action. Although enzymes
`ind mode of catalysis,
`a number al
`sites can be stated
`
`dil cleft formed by ae Lies that Come
`1 aeqquence
`deed,
`residues far apart
`ly
`than adwcent residues in the
`that che graces thé cell he alls
`
`
`
`Engyme alone
`Sr irbe
`and indole
`
`00
`
`Wavelength (rum)
`
`'t:t¢
`
`FIGURE 6.6 Change in spectroscopic
`characteristics with the formation
`of an enryme-substrate comples
`Phares
`me
`nteraty
`of
`th rns
`phaaphain g
`iyplophen
`
`a
`a Tyr #6
`hi
`
`@Sod val 2471
`—s
`Leu 24a
`Camphor (substan)
`
`CHAPTER 0 « Enryren Basic Concept and
`Kinetics
`
`a we
`Phe 87 »
`-
`
`Asp 297
`
`i.
`
`a
`¥
`«
`
`®
`i
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`5. The specificity of binding depends on the precisely defined arrangement of
`atoms in an activ site. Because the enzyme and the substrate interact by
`means of short-range forces that require close contact, a substrate must have
`a matching shape tofit into the site. Emil Fischer's analogy of the lock and
`key (Figure 8.9), expressed in 1890, has proved to be highly stimulating and
`fruitful. However, we now knowthat enzymes are flexible and that the
`shapes of the active sites can be markedly modified by the binding of sub-
`strate, as was postulated by Daniel E. Koshland,Jr., in 1958. The active site
`of some enzymes assume a shape that is complementaryto that of the tran-
`sition state only after the substrate is bound. ‘This process ofdynamic recog-
`nition is called induced fit (Figure 8.10)
`
`concentrations, Before we can accurately interpret
`
`Substrate
`
`+
`
`——"
`
`Enzyme
`
`in thes model, the
`FIGURE 6.10 Induced-fit model of enryme-substrate binding.
`enzyme changes shape on substrate binding. The actwe ste forms a shape complementary
`to
`the substrate only after the substrate has been bound
`
`6.4 THE MICHAELIS-MENTEN MODEL ACCOUNTS
`FOR THE KINETIC PROPERTIES OF MANY ENZYMES
`
`mary function of enzymes 16 to enhance rates of reactions so that
`compatible with the needs ofthe organism. To understand how en-
`are
`they
`1,
`we need a kinetic description oftheir activity. For many en-
`Eyres: fut
`f catalyais V5. which 1s defined as the number of moles of
`zymes, ‘th
`per second, varies with the substrate concentration [5] ma
`product formed
`manner shown in Figure 8.11. The rate of catalysis rises linearly as substrate
`1oenbration imecrea
`incl then begins to level off and approach a maxi-
`mum
`at higher substrate
`
`
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`200
`CHAPTER 8 + Enzymes: Basic Concepts and
`Kinetics
`
`FIGURE 6.9 Lock-and-key model of
`enryme-substrate binding.
`inthis
`model, the active site of the unbound
`enpyme is complementary in shape to
`the substrate.
`
`Enzyme
`
`
`
`
`
`Reactionvelocity(¥,,)—
`
`Substvate comcentration [5
`
`FIGURE 6.11 Michaelit-Menten kinetics
`A plot of the
`reat
`‘
`f
`i
`are tthe
`or a“
`cia aA
`(Ven) is
`approached asym
`Michaelis
`constant
`(x
`concentration yietcing
`
`i
`
`‘
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`
`
`CHAPTER 4+ Engynes: Basic Come epts and
`Kinetics
`
`is that the catalytic rate is equal to the product of
`starting point
`entration of the ES complex and ka
`
`the con
`
`\
`
`4,
`
`|ES
`
`10
`
`Now we need to express [ES] in tertns of known quantities. The rates of for-
`mation and breakdown of ES are given by
`
`Rate of formation of ES=kh, E
`
`Rate of breakdown of ES =
`
`(k_,
`
`+ k.)/ES
`
`In a
`To simplify matters, we will work under the steady-state assumption.
`steady state,
`the concentrations of intermeciiates,
`in this case [ES], stay the
`ame even if
`the concentrations of starting materials and products are
`changing, This occurs when the rates of formation and breakdown ofthe
`ES complex are equal. Setting the right-hand sides of equations 11 and 12
`equal RIVES
`
`h
`
`By rearranging equation 14, we obtain
`
`EIS//ES
`
`ty
`
`fk
`
`(13)
`
`(14)
`
`Equation 14 can be simplified by defining a new constant, Ky), called the
`Michaelis constant
`
`Kyi
`
`:
`k
`
`(15)
`
`Nate that Ky i has the units of concentration kK if 1S an Important charac-
`teristic of enzyme-substrate interactions and is independent of enzyme and
`substrate concentrations
`
`Inserting equation 15 into equation 14 and solving for [ES] yields
`
`(16)
`
`of equation 16. The concentrationof
`Now let us examine the numerator
`in ri br cl substrate || Very nearly equal
`to the
`total substrate con
`centration, provided that
`the concentration of enzyme is much lower than
`that of substrate. The concentration of uncombined enzyme
`[E) is equal
`to the total
`enzyme concentration | E|-- manus the concentration af the ES
`
`i camps x
`
`
`
`
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`CHAPTER 4 = Enzymes: Basic Concepts and
`Kinehes
`
`TABLE 6.5 Ay Values of some enzymes
`
`Enzyme
`
`Substrate
`
`Chymotrypsin
`Lysozyme
`A- Galactosidase
`The mine deaminase
`Carbonee anhydrass
`
`Penicillinase
`Pyruvate carboxylase
`
`Arginine-tR NWA synthetase
`
`tryptophanamuice
`«1
`Acetyl
`Hexa-N-acetylg¢]ucosamine
`Lactose
`Threonine
`CO
`
`Benzylpeniillin
`Pyruvate
`HCO,
`ATP
`
`Arginine
`tRNA
`ATP
`
`S000)
`hy
`4000
`S00)
`A000)
`
`50)
`400)
`joo0
`460)
`
`4
`
`fo
`
`V cx
`
`enzyme i the concentration ofactive sites
`
`equal to the dissociation constant of the ES complex if
`When this condition is met, Ky) is a measure
`omplex:
`a high Ky,
`indicates weak binding; alow
`iding.
`It must be stressed that Ky; indicates the affin-
`hen k_,
`1s much greater than ky
`veals
`the turnover number
`ol an enzyme, ” hich
`ules
`converted inte product by an enzyme mole-
`zyme
`is
`fully saturated with substrate.
`[t is equal
`
`hisalso called &.,,. The maximal rate, Vax
`fan
`
`with the use of « urve-litting Programs on a computer (See the appendix to
`this chapter for alternative means of determining Ky, and V,,,..). The Kay
`values of enzymes range widely (Table 8.5). For most enzymes, Ay, lies be-
`tween 107! and 107°
`M. The Kyy value for an enzyme depends on the pat-
`ticular substrate and on environmental conditions such as pH, temperature,
`and bons strength The Michaelis constant, Kat has Tan meanings First,
`Kyq 18 the concentration of substrate at which half the active sites are filled
`Thus, Ay provides a measure of the substrate concentration required for
`significant catalysis to occur.
`In fact, for many enzymes, experimental evi-
`dence suggests that Ky, provides an approximation of substrate concentra-
`tion in vive. When the Ku is known,
`the fraction of sites filled, fe 3, at any
`substr ale concentration can be 3 alculated fr om
`f
`fo
`oo
`Si + Kay
`
`(24)
`
`Second, Ky; is related to the rate constants of the individualsteps in the
`catalytic ac henne given in equation 9.
`In equation 15 Kay 15 defined as
`(hk
`+
`ky ky. Consider a limiting case in which k_
`|, is much greater than
`Bs, Under such circumstances, the ES complex dissociates to E and 5 much
`more rapidly than product is formed, Under these conditions (k_, >> bo),
`
`The dissociation constant of the ES ¢ omplex Is given DY
`
`(25)
`
`26)
`
`
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`206 |
`
`CHAPTER 8 + Enzymes: Basic Concepts and
`Kinetics
`
`values for several different substrates of chymotrypsin (Section 9.1.1). Chy
`motrypsin clearly has a preference for cleaving next to bulky, hydrophobic
`side chains
`How efficient can an enzyme be? We can approach this question by de-
`termining whether there are any physical limits on the value of &.,./ Kay.
`Note that this ratio depends on k,, k_,, and k;, as can be shown bysubsti-
`tuting for Ky,
`
`hea
`Fan
`
`kh, <
`Row
`Fea
`ig ae Okee
`
`(30
`
`)
`
`The whlizahon of attractive forces to
`lure a substrate into a site in which
`
`it undergoes a transformation of
`structure, at defied by Wilkam P
`Jencks, an enzymologist, who coined
`the term
`
`A goddess of Greek mythology, Circe
`lured Cehysseus's men to her house
`and then tunsionmed them into pigs
`
`PLLC anhwdrase
`
`Suppose that the rate of formation of product (hk...) is much faster than the
`rate of dissociation of the ES complex (k_,). The value of k../Kyq then
`approaches &). Thus, the ultimate limit on the value of k../ Kay is set
`by &,, the rate of formation of the ES complex. This rate cannot be faster
`than the diffusion -contralled encounter af an enzyme and tts substrate Duffu-
`sion limits the value of ky &O) that it cannot he higher than between id? and
`10° s°' M~'. Hence, the upper limit on k,,,/Ky,
`is between 10° and
`10° a
`'M
`ratios of the enzymes superoxide dismutase, acetyl-
`The Ri Ky,
`cholinesterase, and triosephosphate isomerase are between 10° and 10" '
`Mo! Enzymes such as these that have k.,,/A), ratios at the upper limits
`have attained kinetic perfection, Their catalytic velocity is restricted only by
`the rate at which they encounter substrate in the solution (Table 8.8), Anyfur-
`ther gain in catalytic rate can come only by decreasing the time for diffu-
`sion. Rernember that the active site is only a small part of the total enzyme
`structure. Yet, for catalytically perfect enzymes, every encounter between
`enzyme and substrate is productive.
`In these cases, there may be attractive
`electrostatic forces on the enzymethatentice the substrate to the activesite.
`Chese forces are sometimes referred to poetically as Circe effects.
`The limit imposed by therate of diffusion in solution can also be partly
`overcome by confining substrates and products in thelimited volume of a
`multienzyme complex. Indeed, some series of enzymes are associated into
`organized assemblies (Section 17.1.9) so that the product of one enzyme is
`very rapidly found bythe next enzyme.
`In effect, products are channeled
`from one enzyme to the next, much as in an assembly line.
`
`TABLE 8.8 Enzymes for which k,,./Ky is close to the diffusion-controfied
`rate of encounter
`
`Enzyme
`Acetyicholinesteraze
`
`(a ks
`
`Bear! Kya (5 "MO
`
`
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`8.5 ENZYMES CAN BE INHIBITED
`BY SPECIFIC MOLECULES
`
`209
`
`Enaymne bnhebiion
`
`Subvirate
`
`The activity of many enzymes can be inhibited by the binding of specific
`small molecules and ions: This means of inhibiting enzymeactivity serves
`a8 a Major control mechanism in biological systems. The regulation of al
`hostenc enzymes typifies this type of control.
`In addition, many drugs and
`toxic agents act by inhibiting enzymes.
`Inhibition by particular chemicals
`can be a source of inghit inte the mechaniam of enzvme action specitic in-
`hibitors can often be used to identify residues critical for catalysis. The value
`of transition-state analogs as potent inhibitors will be discussed shortly
`Enzyme inhibition can bee either re versible of iffewv eruble An Fret ersible
`inhibitor dissociates very slowly from its target enzyme because it has be
`come tightly bound to the enzyme, esther covalently or noncovalently. Some
`imreversible inhibitors are important drugs. Penicillin acts by covalently
`modifying the enzyme transpepticlase, thereby preventing the synthesis of
`bacterial cell walls and thus killing the bacteria (Section 8.5.5 Aspirin acts
`by covalently modifying the enzyme cyclooxygenase, reducing the syn
`thesis of inflammatory signals
`Reversible mhibition,
`in contrast with irreversible inhibition, is charac-
`tenzed by a rapid dissociation of the enzyme—inhibitor complex.
`In com
`1
`tnfabition, ar enzyme can bind substrate (forming an ES complex)
`or inhibiter (EI) but not both (EST). The competitive inhibitor resembles
`the substrate and binds to the active site of the enzyme(Figure 8.15). The
`substrate ia thereby prevented from binding to the same active site, A com
`petitive inhibitor diminishes the rate of catalysis by reducing the proportion of
`enzyme molecules bound to w substrate. At any given inhibitor concentration,
`Competitive inhibition can be reliewed by increasing the substrate concen
`tration. Under these conditions, the substrate “outcompetes” the inhibitor
`for the active site. Methotrexate is a structural analog oftetrahydrofolate,
`a coenzyme for the enzyme dihydrofolate reductase, which plays a rolein
`the biosynthesis ot purines and pyrimidines (Figure 8.16). Tt binds to di-
`hydrofolate reductase 1000-fold more tightly than the natural substrate and
`inhibits nucleotide base aynthesis.
`It is used to treat cancer
`
`Irom bundling
`
`moeliti
`ipeetit
`(lap) enryrne— substrate
`inhibitor,
`(meddle) a cortpetitieve mbsbrtor
`complies,
`bers at the actwe site and thus prevents
`the substrate fram binding:
`(bottam) a
`Toroerpetitre nhbetor coe,
`re
`t prevent
`f
`the gubbstrate
`
`HM
`
`Tetvabydrotodate
`
`
`
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`Why ts penicillin such an effective inhibitor of the transpeptidase? The
`highly strained,
`four-membered f-lactam ring of penicillin makes it
`especially reactive, On binding to the transpeptidase, the serine residue at
`the active site attacks the carbonyl carbon atom of the lactam ring to form
`the penicilloyl-serine derivative (Figure 4.31). Because the peptidase par-
`hicipates in ite own mactivation, penicillin acts as a suicide inhibitor
`
`‘il ear in il hi mW int fegra a
`
`Coenzyme
`
`lypical reaction type
`
`Consequences of deficiency
`
`Thiamine pyrophosphate
`
`Aldehyde transfer
`
`Flavin adenine
`hinucleotide (FADD)
`
`Pyridoxal ph epihats
`
`Ohedahianreductian
`
`(aroup trainster to.or from
`immuno acids
`( hidation reduction
`
`group transfer
`ATT dependent
`arbosxylateen and
`l-froup transter
`1
`one carbon
`nite:
`thymine
`
`Beribers (weight loss,
`heart problema,
`neurological
`dyatunction |
`(Cheleosis and angular
`stomatitus (leaons of the
`mouth), dermatitis
`
`Depresnion, corbin
`convulsions
`Pellagra (dermaritis
`lepreasion,
`diarrhea)
`Hypertension
`Rash about the
`eyebrows, muscle pan
`fatigue (rare
`Anomia, nctinal- tube
`lefects in development
`
`Ancmia, Pernecivus
`inca, Methylene
`
`(ewollen ane
`Scurvy
`bles Lng Gums
`
`
`
`216)
`CHAPTER 8+ Entymes; Bast Concepts and
`Kinetecs
`
`FIGURE 8.31 Formation of a
`penicilloyl-enzyme compiles. Penicifin
`heacts with the transpeptidaw to form
`an inachve comptes, whech is
`indelnetety
`Mable
`
`Glycopeptide
`Iransreplidane
`
`Punicilloyl-oncyes comple
`(enrymatcally inacteen )
`
`8.6 VITAMINS ARE OFTEN PRECURSORS TO COENZYMES
`
`me Earlier (Section 8.1.1), we considered thefact that many enzymes re
`"quire cofactors to be catalytically active. One class ofthese cofactors,
`ter med COCnFYMes, OOnssts of smal] Ofganit molecules, mary of which ane
`derived from vitamins. Vitamins themselves are organic molecules that are
`needed in small amounts in the diets of some higher animals. These mole+
`
`-—
`TABLE 6.9 Water-Soluble Vitamins
`a
`
`Vitamin
`
`Thiamine (i
`
`
`
`
`
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`Proly! inpdroaplase
`+ moobete
`
`OH
`a-Hydiroxyproly!
`reudue
`
`Proline ns hydroxylated at C-4 by the
`FIGURE 0.55 Formation of 4-hydronyproline.
`acton of profyl hydrorylase. an enzyme that actwates molecular oxygen
`
`tic pe ly peptides have been
`
`still required for the continued activity of proyl hydroxylase. This enzyme
`synthesizes 4-hydroxyproline, an amino acid that ts required in collagen,
`the major connective tissue in vertebrates, but is rarely found anywhere else.
`How is this unusual amino acid formed and what ts its role? The results of
`radioactive-labeling studies showedthat proline residues on the amino aide
`of glycine residues in nascent collagen chains become hydroxylated. The
`oxygen atom that becomes attached to C-4 of proline comes from