2.

Enzimi:
concetti fondamentali e
meccanismo di catalisi
enzimatica

1
 Proprieta
Catalizzatori dei sistemi biologici
Normalmente alta specificit verso il substrato
Condizioni di reazione blande
Soluzioni acquose
Temp. non elevate (25-37C)
pH neutro
Nella cellula agiscono in sequenze ordinate
Molto importanti:
Causa di patologie
Strumento di diagnosi di molte malattie
Applicazioni in chimica industriale (detersivi, biocatalisi etc.)
Applicazioni nell industria alimentare (fermentazione, lievitazione )
Applicazioni in biosensori
Prof. G. Gilardi - Biological Chemistry
 Gli enzimi sono proteine
Possono funzionare grazie ai gruppi reattivi delle
catene laterali degli aminoacidi
Possono necessitare
COFATTORI = ioni inorganici
COENZIMI = molecole organiche o metallo-
organiche, per es vitamine
GRUPPI PROSTETICI = coenzimi legati
covalentemente agli enzimi
OLOENZIMA = enzima con il suo cofattore o
coenzima o gruppo prostetico
APOENZIMA = sola parte proteica di un enzima
Prof. G. Gilardi - Biological Chemistry
Esempi di cofattori

Prof. G. Gilardi - Biological Chemistry

Esempi di coenzimi

Prof. G. Gilardi - Biological Chemistry

Riconoscimento*specico**
del*sito*a0vo*

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A3enzione*alla*dinamica*molecolare:*

Prof. G. Gilardi - Biological Chemistry!

 Specicit*dei*si9:**
dierenze*dovute*allisomeria*o0ca:*
Isomeri ottici!

Menta**** * * * *** * * * *Cumino**

8!
 Isomeri ottici!

dolce* * * * *** * * * *****amaro*

9!
 .*Basi*del*processo*visivo*nellocchio.*
Isomeri geometrici!

Retinale (vitamina A) legato alla proteina opsina = rodopsina. La luce converte il cis-retinale
in trans-retinale che cambia conformazione della proteina che inizia un processo che porta10!
alla
generazione di un impulso nervoso che va al cervello !
Classificazione

Prof. G. Gilardi - Biological Chemistry

 The role of enzymes
Biological processes must be carried out in short time-
scales: the human nerve cell must respond instantly to
stimulus, bacteria must reproduce in 20 min
Reactions must be quickly carried out: reactions that
takes place in timescale of hours are useless to life
Enzymes are catalysts: they speed the velocity of a
reaction without taking part to it
Their activity can be regulated depending on the cellular
needs

Prof. G. Gilardi - Biological Chemistry

THERMODYNAMICS AND
KINETICS OF ENZYME
CATALYSIS

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 Transition states and reaction rates

Thermodynamics*

Kine9cs*

Prof. G. Gilardi - Biological Chemistry

Prof. G. Gilardi - Biological Chemistry
 Relationship between k and Ea: the
Arrhenius equation

A useful generalization supported by the

Arrhenius equation is that, for many common
chemical reactions at room temperature, the
reaction rate doubles for every 10 degree
Celsius increase in temperature.

Prof. G. Gilardi - Biological Chemistry

Role of a catalyst

Prof. G. Gilardi - Biological Chemistry

Prof. G. Gilardi - Biological Chemistry
Risultato*della*catalisi*enzima9ca*

19!
Prof. G. Gilardi - Biological Chemistry
Prof. G. Gilardi - Biological Chemistry
Enzymes have an optimal pH and
temperature for activity

Prof. G. Gilardi - Biological Chemistry

Specificity-complementarity of
the enzyme active site

What is the enzymes active site

complementary to?

Prof. G. Gilardi - Biological Chemistry!

 NADP+!

Tetraidrofolato !

Diidrofolato reduttasi! Prof. G. Gilardi - Biological Chemistry

 NADP+!

Tetraidrofolato !

Diidrofolato reduttasi! Prof. G. Gilardi - Biological Chemistry

Prof. G. Gilardi - Biological Chemistry
Prof. G. Gilardi - Biological Chemistry
Amminoacidi*della*catalisi*acidoFbase*
pKA

3.5 - 4.5
10.5 12.5
8.2

6.0

.
10.1

Gruppi*funzionali*di*amminoacidi*contenu9*nel*sito*a0vo*di*enzimi*per*la*
partecipazione*nella*catalisi*come*donatori*o*acce3ori*di*protoni.*La*loro*pKA*
pu*essere*fortemente*inuenzata*dal*microambiente*del*sito*catali9co
Prof. G. Gilardi - Biological Chemistry! *
 Enzyme kinetics:
The Michaelis-Menten model

Prof. G. Gilardi - Biological Chemistry!

 Michaelis-Menten kinetics
For a simple reaction:

! Let s make the following assumptions:

! The reverse reaction E + P > ES (governed by k-2) is negligible
! The reaction that gives the product is 1st-order, that means it depends
only on [ES]:

! If we know k2 and [ES] we can the calculate the velocity v. But in an

experiment we only know the total conc. of enzyme [E]t and the conc. of
substrate [S] that we add into the reaction mixture. We therefore must
elaborate a theory that allows to calculate v from [E]t and [S]

! We*know*that:*
Prof. G. Gilardi - Biological Chemistry
 The steady-state hypothesis
In 1925 Briggs and Haldane
proposed the steady-state
hypothesis that states: the
more ES is formed, the more
it is consumed to give E + P.
Therefore when a reaction is
started by mixing E and S,
the complex ES will quickly
build up till it reaches a
constant conc. that remains
the same untill all S is
consumes.

Prof. G. Gilardi - Biological Chemistry

 In the steady-state, the rate of ES formation and ES breakdown are
equal, therefore:

! Rearranging:*

! The*deni9on*of*the*MichaelisFMenten*constant*is*:*

! Therefore:*

From here we see that KM has the units of concentration

Prof. G. Gilardi - Biological Chemistry
 We*recall*that:*

Subs9tu9ng:*

In this way we have defined [ES] as a function of [E]t e di [S] that are
2 parameter that we set in the reaction and we know
Prof. G. Gilardi - Biological Chemistry
 Going*back*to*the*ini9al*problem/ques9on:*

Subs9tu9ng:*

This is the Michaelis-Menten equation that describes enzyme

kinetics. Note 2 key points:
KM is a ratio of rate constants for a specific reaction, and it is
therefore characteristic for the reaction considered
KM has the units of concentration in the range of mM or M

Prof. G. Gilardi - Biological Chemistry

The Michaelis-Menten graph

Prof. G. Gilardi - Biological Chemistry

Velocit*iniziale*di*una*reazione*enzima9ca*

Si*determina*la*velocit*iniziale*v0*dalla*pendenza*della*tangente*alla*curva*
che*passa*per*il*tempo*0.*Per*ogni*concentrazione*di*substrato*la*velocit*
della*reazione*diminuisce*in*funzione*della*trasformazione*SF>P.
Prof. G. Gilardi - Biological Chemistry!
*
 Dipendenza*della*v0*dalla*conc.*di*S*

v0 = vmax[S]/(KM+[S]) !

Quando*[S]**bassa,*si*ha*KM>>[S]*e*il*termine*[S]*al*denominatore*di*v0*=*vmax[S]/(KM+[S])*
diventa*irrilevante*per*cui*lequazione*si*semplica*in*v0*=*vmax[S]/KM*
Quando*[S]**alta,*si*ha*[S]*>>KM**e*il*termine*KM*al*denominatore*di*v0*=*vmax[S]/(KM+[S])*
diventa*irrilevante*per*cui*lequazione*si*semplica*in*v0*=*vmax*
 From the graph it follows that when [S] >> KM, the reaction
approaches vmax - in this situation all the enzyme
molecules are saturated with the substrate.
Therefore:

! Then:*

! Therefore:* This*is*the*most*
commonly*used*
MichaelisFMenten*
Prof. G. Gilardi - Biological Chemistry equa9on*
 Reaction rates for multi-step
reactions
Lets consider the multi-step reaction:

! It also can be described by the Michaelis-Menten equation, but k2

must be replaced by a more general rate constant kcat:

! The rate constant kcat incorporated all the rate constants for all the
steps between ES and E + P, while for the standard 2 steps reaction,
kcat=k2 but as kcat is a more general terms is more correct to always
use this parameter
! It follows that:

Prof. G. Gilardi - Biological Chemistry

 Definitions of kcat , KM , kcat/KM
KM is the measure of the substrate conc. necessary for
effective catalysis.
KM can be defined as the affinity of the E for S: low KM = high
affinity, high KM = low affinity (see graph).
An enzyme with high KM necessitates a higher conc. of substrate
to achieve a given reaction velocity compared with an enzyme
with a low KM , when kcat is the same.
kcat measures the generation of product under optimum
conditions saturated enzyme
kcat measures the number of substrate molecules turned over
(transformed into product) per enzyme molecule per seconds. Its
units are s-1. For this reason kcat is often called turnover number.
kcat/KM measures the enzyme efficiency
Prof. G. Gilardi - Biological Chemistry
Examples of kcat , KM , kcat/KM

Prof. G. Gilardi - Biological Chemistry

 Maximum value for kcat/KM
kcat / KM corresponds to the 2nd-order rate constant of the E + S
combination;
This 2nd-order rate constant has a theoretical maximum value: this is
determined by the frequency at which E and S can collide
A reaction that occurs at this velocity is said to be diffusion limited
A diffusion limited reaction is a reaction for which at every possible
molecular encounter leads to a product the molecular encounters
therefore is the only limit to the reaction
It has been calculated that such reaction depends on parameters of
the diffusion theory, and the maximal value is 108-109 M-1s-1
An enzyme with kcat / KM in the range of 108-109 M-1s-1 is a perfect
enzyme. Examples are the Fumarase and even better the Triose
Phosphate Isomerase

Prof. G. Gilardi - Biological Chemistry

Measure of substrate preference

Prof. G. Gilardi - Biological Chemistry

 Experimental measurements
of KM, vmax and kcat
Usually this is done by using
graphical representations of
equations that produce linear
graphs
One method is the double
reciprocal plot also called
Lineweaver-Burk plot

Prof. G. Gilardi - Biological Chemistry

 Another method is
that called Eadie-
Hofstee plot:

Prof. G. Gilardi - Biological Chemistry

 Multi-substrate reactions
More complex enzyme catalyzed reactions may involve more than
one substrate or more than one product. One example is:

The possible cases are

Random substrate binding

Ordered substrate binding

Ping-pong mechanism

Prof. G. Gilardi - Biological Chemistry

Enzyme Inhibition

Prof. G. Gilardi - Biological Chemistry

 Types of inhibition
Reversible inhibition:
When the I is non-covalently bound to the E
and it can be removed by displacement
It can be:
Competitive inhibition
Non-competitive inhibition
Mixed competitive and non-competitive
Irreversible inhibition:
When the I is covalently bound to the E
Prof. G. Gilardi - Biological Chemistry
 Reversible inhibition: Competitive

Here I competes with the S for the same

binding site. It increases the apparent KM
(Kmapp)

KI = dissoc. const.
for I binding

KI = [E][I] / [EI]
Prof. G. Gilardi - Biological Chemistry
where [I] = free I
 Inibitori reversibili -
competitivi
Queste molecole di solito assomigliano al S e competono
con esso per il sito attivo formano il complesso EI.
Aumentando la [S] l effetto dell inibitore diminuisce, la
vmax verra raggiunta a KM piu alte. Dunque la KM sara
una Kmapp e sara tanto piu alta quanto piu alta e la [I].

Prof. G. Gilardi - Biological Chemistry!

Here:*
*
v0*=*vmax[S]*/*KM+[S]*
*
= 1+[I]/KI!
*
KI = [E][I]/[EI]!
*
Here KM = Kmapp that is (1 + [I] / KI)
Increasing [I] increases the KMapp
If [S] becomes very large, it will outcompete [I]
vmax and hence kcat is unchanged as vmax = kcat [E]t
Prof. G. Gilardi - Biological Chemistry
Prof. G. Gilardi - Biological Chemistry
Prof. G. Gilardi - Biological Chemistry!
 Inibizione
competitiva
Vmax invariata
KM aumenta con [I]

Prof. G. Gilardi - Biological Chemistry!

 An example of
competitive inhibitor

Prof. G. Gilardi - Biological Chemistry

 Inibizione reversibile:
inibizione incompetitiva

I si lega ad un sito diverso

da quello del S, ma si lega
solo a ES (cioe quando S
e presente)
In questo caso sia vmax che
KM sono diminuite

v0*=*vmax[S]*/*KM+[S]*
*
*=*1+[I]/KI*
*
KI*=*[ES][I]/[ESI]*
Prof. G. Gilardi - Biological Chemistry!
 Inibizione
incompetitiva

! Vmax e KM
diminuiscono con [I]

Prof. G. Gilardi - Biological Chemistry!

 Inibitori reversibili -
misti o non-competitivi
In questo caso I si lega
ad un suo sito distinto
da quello del S.
La KM non varia in
quanto il sito del S non
e stato influenzato
I si lega, sia che ci sia S
o meno, cioe sia a E
che a ES
Quando si lega riduce la
[Etot] attivo. Siccome
vmax = kcat [Etot], allora la
vmax diminuisce
Prof. G. Gilardi - Biological Chemistry!
 The Kmapp is not influenced by the I
The kcatapp decreases with increase of I
Prof. G. Gilardi - Biological Chemistry
 Inibizione non-competitiva
Prof. G. Gilardi - Biological Chemistry
 Inibizione
non-
competitiva
Vmax diminuisce con [I]

KM invariata

Prof. G. Gilardi - Biological Chemistry!

Inibizione mista

Prof. G. Gilardi - Biological Chemistry!

 Irreversible inhibition

Here I binds covalently the E

These I are toxic compounds
Some irreversible inhibitor mimic
the transition state analog (see for
example DFP and sarin)
In some cases the E bind the I, it
metabolizes it to its P that then
binds covalently the E. These are Diidopropyl*uorophosphato,*DFP,*
called suicide inhibitors reacts*with*a*Ser*of*the*protein,**
that*can*be*a*serineFprotease**
or*acetylcholinesterase*
to*form*a*covalent*adduct*that*
blocks*the*enzyme*
Prof. G. Gilardi - Biological Chemistry
Prof. G. Gilardi - Biological Chemistry
Irreversible*
enzyme*
inhibitors*

Prof. G. Gilardi - Biological Chemistry

Prof. G. Gilardi - Biological Chemistry!
Prof. G. Gilardi - Biological Chemistry!
Prof. G. Gilardi - Biological Chemistry!
Indinavir!
!
!
!
!
Nelfinavir!
!
!
!
Lopinavir!
!
!
!
Saquinavir!

Prof. G. Gilardi - Biological Chemistry!

Regolazione*enzima9ca*

Prof. G. Gilardi - Biological Chemistry!

 Enzimi*regolatori,**
regolazione*allosterica*e*modicazione*covalente*

Nel*metabolismo*cellulare*deve*esserci*la*possibilit*di*modulare*
favorendo*o*sfavorendo*una*determinata*sequenza*di*reazioni*a*seconda*
del*fabbisogno*della*cellula*
La*gran*maggioranza*di*una*via*metabolica*consiste*in*sistemi*mul9F
enzima9ci*che*funzionano*a*catena*in*modo*sequenziale,*e*il*minor*
dispendio*energe9co*si*ha*quando*il*primo*enzima*della*via*metabolica**
un*enzima*regolatore.*
Esistono*due*classi*di*enzimi*regolatori*che*seguono*due*meccanismi:*
Modulazione*allosterica*non*covalente*
Modicazione*covalente*reversibile*
Un*enzima*regolatore*in*genere**sensibile*ad*una*molecola*che*si*chiama*
modulatore*e*il*processo*avviene*grazie*alla*regolazione*allosterica**
Entrambe*le*classi*sono*in*genere*enzimi*a*pi*subunit*e*il*sito*catali9co*e*
quello*regolatore*si*trovano*su*subunit*dis9nte*

Prof. G. Gilardi - Biological Chemistry!

 Enzimi*allosterici*e*modulatori*
Gli*enzimi*allosterici*hanno*in*genere*un*
sito*catali9co*per*il*substrato*su*una*
subunit*de3a*catali9ca*e*un*sito*di*
legame*per*il*modulatore*su*una*seconda*
subunit*de3a*regolatrice.*
Come*gi*visto*per*lemoglobina,*le*
diverse*subunit*si*inuenzano*a*vicenda,*
il*legame*di*un*modulatore*posi9vo*pu*
cambiare*la*conformazione*del*sito*a0vo*
per*dare*un*enzima*pi*a0vo*
I*modulatori*possono*essere*posi9vi*o*
nega9vi;*ques9*ul9mi*non*devono*essere*
confusi*con*gli*inibitori*(anche*se*spesso*
viene*chiamata*inibizione**v.*sigmoide)*
In*genere*si*indicano*con:*
Regolazione*posi9va:*
Regolazione*nega9va:**
Prof. G. Gilardi - Biological Chemistry!
 Tipico*esempio:**
inibizione*o*modulazione*
retroa0va*(a*feed*back*

Spesso*il*prodo3o*ul9mo*di*
una*via*metabolica**un*
modulatore*nega9vo*del*
primo*enzima*della*via*stessa*
Vedi*esempio*della*sintesi*
della*isoleucina*dalla*
treonina*
*

Prof. G. Gilardi - Biological Chemistry!

 Comportamento*di*enzimi*
allosterici:*sigmoide*
La*relazione*tra*v0*e*[S]*negli*enzimi*allosterici*non*obbediscono*alla*cine9ca*
iperbolica*descri3a*da*MichaelisFMenten*
Mostrano*saturazione*solo*a*conc.*di*[S]*elevate,*invece*di*KM*si*parla*di*K0.5*e*di*
[S]0.5*per*indicare*la*conc.*di*S*per*avere*met*della*vmax*
La*cine9ca*sigmoidale*so3ende*interazioni*regolatrici*non*covalen9*tra*pi*
subunit*
Enzimi*allosterici*omotropici:*substrato*e**
*modulatore*sono*iden9ci.*Una*molecola*
*si*lega*al*sito*a0vo*mentre*laltra*si*lega**
*al*sito*regolatore*che*si*trova*su*una**
*seconda*subunita*F>*sigmoide*
Enzimi*allosterici*eterotropici:*il*regolatore*
**un*metabolita,*non*il*substrato*
Prof. G. Gilardi - Biological Chemistry!
Esempi*di*ee0*della*regolazione*
allosterica*sulla0vit*(K0.5*e*vmax):*

Prof. G. Gilardi - Biological Chemistry!

 Modicazione*covalente*reversibile*
Mol9*sono*i*
meccanimi*di*
modicazione*
covalente*reversibile,*
ma*la*fosforilazione**
quella*pi*importante*
Un*terzo*delle*
proteine*eucario9che*
sono*fosforilate*
La*fosforilazione*
avviene*su*1*o*pi*
sequenze*consenso*
riconosciute*da*
proteine*chinasi*
(inserimento)*o*
fosfatasi*(rimozione)*

Prof. G. Gilardi - Biological Chemistry!

 A0vazione*per*scissione*proteoli9ca*
Si*tra3a*di*un*precursore*
enzima9co*ina0vo*che*viene*
a0vato*per*proteolisi*
Nel*caso*di*proteasi*si*parla*di*
precursore*zimogeno,*per*esempio*
chimotripsinogeno*e*chimotripsina*
danno*chimotripsina*e*tripsina*
In*altri*casi*si*parla*di*proFproteine*o*
proFenzimi*(per*esempio*proFinsulina*
e*insulina)*

Prof. G. Gilardi - Biological Chemistry!

 Concepts*to*catch:*
1. Descrivere*con*luso*di*graci*e*formule,*il*ruolo*del*sito*a0vo*di*un*
enzima*nel*processo*della*catalisi*enzima9ca*secondo*la3uale*teoria*che*
coinvolge*uno*stato*di*transizione.!
2. Dimostrare*lequazione*di*MichaelisFMenten*denendo*in*termini*sia*di*
equazione*che*di*graco*e*di*conce3o*la*KM*,*la*vmax,*la*kcat*e*denire*
lecienza*enzima9ca!
3. Descrivi*con*luso*di*formule*e*graci*i*processi*di*inibizione*enzima9ca,*
sia*nonFreversibile*che*reversibile*in*tu3e*le*sue*forme,*con*par9colare*
riferimento*alla*loro*inuenza*sulla*KM*e*kcat***
4. Descrivere*un*9po*di*inibizione*enzima9ca*illustrando*gracamente*gli*
ee0*osserva9*sulla*velocita*massima*di*reazione*(vmax)*e*sulla*costante*
di*Michaelis*Menten*(KM).*
5. Descrivi*con*luso*di*formule*e*graci*i*processi*di*regolazione*allosterica*

Prof. G. Gilardi - Biological Chemistry!