Biochem [Enzymes]
description
Transcript of Biochem [Enzymes]
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Advantages of biocatalysis1. Enzymes are efficient catalysts - to increase the reaction rate of at least 106. ln
their absence . the reaction rate in biological systems is insignificant.
2. Enzymes are characterized by high specificity.
3. Enzymes operate under mild conditions, i.e,a.'atmosPhericPressure;b. in most cases, in the temperature range 20 - 40 oC;c. at pH close to neutral;
4. The reactions proceed in the aqueous media.5. Wastes from the reaction are less harmful to the environment.
6. Enrymatic reactions carried out in a simple apparatus.
7. Enzymatic reactions are characterized by high efficiency.
8. The enzyme catallzes no side reactions.
Types of enzyme selectivitY
1. Ghemoselectivity (e. g.The enzyme hydrolyzes the esters and not hydrolyzesacetals or amides).
2. Regiosetectivity and diastereoselectivity (For example: with many of the samefunitional groups, located in the substrate, enzyme reacts with only one -located in given Place)
3. Enantioselectivity (e.g. the enzyme catalyzes only the reactions of L'amino acidor only the D.saccharide - it is intact to D-amino acid or only the L'saccharide ).
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Enzyrne active siteThe active site is part of an enzyme where substrates bind and undergo a chemical reaction. Theactive site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residuesthat participate in recognition of the substrate. Residues that directty participate in the catalyticreaction mechanism are called active site residues.There are two proposed models of how enzymes work: the lock and key model and the inducedfit model. The lock and key model assumes that the active site is a perfect fit for a specificsubstrate and that once the substrate binds to the enzyme no further modification is necessary.The induced fit model is a development of the lock-and-key model and instead assumes that anactive site is more flexible and that the presence of certain residues (amino acids) in the activesite will cause the enryme to locate the conect substrate, after which conformational changesmay occur as the substrate is bound.
E r-r:;\irlr: ,: ltatr,il::; slr;r;:,*sliqlrtly er :r-rl-,stret* liirrils
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lnduced fit model
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-$ubstrate
EnzyrneEnzyrne -SubstrateComplex
lnduced-lit Modet. - The enzyme active site forms a complemehtaryshape to the strbstah after binding,
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NtlL\l L/lmportance of complex: enzyme:substrate
ln the enryme's active centerthe are: substrate binding site and the catalytic site. Reaction canrun when the substrate and enzyme are complementary to each other. For example, enrymatichydrolysis of acetylcholine catalyzed by acetylcholine esterase.
ACETYLCHOLINE
H2 CHz
N*- trimethyl groupis bonded to enzyrnein substrate bonding site
Ester bond is directedto catalytic site of esterase
Breakdovun of ester bond by enzyme aetylct'toline esferase leads totrydrolysb of acetylcholineto choline and acetic acld
Ai
k\
HsCwHscl
oilo-c
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lmportance of complex: enzyme:substrateln the enzyme's active center the are: substrate binding site and the catalytic site. Reaction can
run wtrenihe substrate and enzyme are complementaryto each other. For example, enrymatic
hydrolysis of acetylcholine catalyzed by acetylcholine esterase.
ACETYLCHOLINE
Hz-CHz
N*- trimethyl groupis bonded to enzymein substrate bonding site
Ester bond is directedto catalytic site of esterase
Breakdorn of ester bond by enryme acetytdtoline esterase bads totrydrolysis of acetylcholineto choline and acetlc acid
Enzyme active site charasteristics:1.
-Substrates bind to the active site of the enzyme or a specificity pocket through hydrogen
bonds, hydrophobic interactions, temporary covalent inter:actions (van der Waals) or acombinaiion of all of these to form the enryme'substrate complex.
2. Enryme active site has a form of pocket or slot in the protein structure.3. An active site occupies a relativeiy small space of the enzyme molecule. A few of amino acid
residues of enryme come into direct contact with the substrate molecule.l. Amino acid residues, having the acceptor and donor groups in their aliphatic side chain,participate in formation of enryme active site'
5. Aminoacid residues forming {he enzyme active site will act as donors or acceptors of protonsor other groups on the subsirate to ficilitate the reaction. The active site modifies the reactionmechaniim in orderto change the activation energy of the reaction'
6. The product is usually unstalb in the active site due to steric hindrances that force it to bereleased and retum the enzyme to ib initial unbound state.
HsCy,HscT
HsC
oil
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IEC Clssstfication of Enzymesl. Oxidases or Dehydrogenases
They c at alyz e the oxidati on-re duct ion re act ions
2.Transferases
They transfer offunctional groups (8.i. aldehyde, ketone, and acyl groups)3.Hydrolases
They catalyze the hydrolysis reactions @or example. hydrlolysis of peptides, esters,amides)
4. Lyases
They catalyze the addition to double bonds or its reverse
S.Isomerases
They catalyze the isomerization reactions ( racemization, multi bonds transfer,transformation of functional groups)
6.Ligases or Synthetases
They catalyze theformation of bonds withATP cleavage (C - C, C- heteroatom bondsfortration)
An international code for the enzyme
International code forthe enryme consists of two letters and four numbers sepamted byperiods. His schedule is as follor,vs: EC a.b.c.d.Symbol EC (enryme code) means that the number of international concem afterthe genetic code.EC
- means Enzyme Commission.
The number a indicates the number of a class of enryme;Number of b- indicates the number subclass within the class;The number c represents the number of subclass within subclass;The number d represenh the number of d-enryme within the previously mentioned subclass.
To each wetl known enzyme is assigned a unique identification number.
Forexample, lactate dehydrogenase has EC1.1.1.27 code number.The first number ({) means thatthe enzyme belongs to 1 class, so it is oxidoreductase. Thesecond number (1) means thatthe enryme belongs to a subclass, which includes all ofoxidoreductases, which disconnect a pair of hydrogen atoms from a group of HC-OH. The thirdnumber (1) indicates that this enzyme belongs to subclass 1, including all enrymes, which transferthe hydrogens disconnected from the HC-OH group on the NAD*. The fourth number (27) is thenumber assigned to this enryme within subclass EC.1.1.1.
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Application of enzymes in medical practice
Enrymes have numerous practical applications in the laboratory diagnosis of many diseases
and are laboratory reagents and drugs'Enryr.. ar. used to rlepair the genJtic material of cells. This opens up new opportunities forthe ireatment of congenital metabolic diseases and cancer.Enrymes selve as markers of disease'The activity of some enzymes in tissues and body fluids change during the course of various
l::ffi;pe, transaminase level in blood plasma increases during the course of myocardialinfarction or liver damage.Amylase. an enryme that breaks down starch - is increased in patients with pancreatitis'
oiainostic signifi-cance has also relationship between the activity of some specific isoenrymes'
So one:- ttealthy people have lower activity of LDH-I (tactate dehydrogenasel) than LDH'2 in plasma'-
patienG-with myocardial infarction have LDH'1 greaterthan LDH'2'
Enzymes as drugs and reagents
Some enzymes are used as drugs:Lipase. an enzyme hydrolyzinifrtr. is useful in treating deficiencies in secre{ion of thepancreas.
. ..!-4l^a__r_Asparaginase - an enzyme that breaks down asparagine ' is useful in the treatment of
leukemias.
some enzymes are used as reagents in laboratory practice, e'9.Urease cin be used to determine concentration of ureaLactate Dehydrogenase - forthe determination of lactate.
Enrymes in biotechnology and gene therapy
using the nucleases one can cut,,wrong" sections _of DNAand fulfillthese
places by
"gooi" sections derived from the healthy cells of th9_s-11e species.
- .. r
Anastomosis (theconnection of two stiuctures) of DNAfragments is possible due to DNAligases.ifiii ,.r.r possible to repair the damaged gene, and opens up the prospect of developing anewfield of medicine known as gene therapy'
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OxireductasesOxireductases catalyse the oxidation and reduction reactions. An example may be belactate dehydrogenase, which converts lactate to pyruvate and vice versa withparticipation of NAD*.
NAD* NADH + H*
?n,lactatedehydrogenase
?H,c:oI
coo 'o-
pyruvate
H-C-OHI
cc;' 'o-
lactate
/ l''
Transferases
Transferases transfer chemical groups from the substrate (the donor)for a product (recipient or acceptor). For example, alanine aminetransfelasetransfers NH, group from glutamate to pyruvate; as the products alanine anda-ketogli,rtarate are formed. PLP is a coenzyme in this reaction.
coo'I
?H,QHzlr
H-?-NHa'coo-
glutamate
QHsIc:oIc99-
pyruvate
alanineaminotransferaza
coo-I
CH,l-GH,l-c:o
I
coo'
a-ketoglutarate
QHsH-?-NH3.
coo'
alanine
k /r,
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Lyases
A tyase is an enryme that catalyzes the breaking of various chemical bonds by means ot!.tthan hydrolysis and oxidation, often forming a new double bond or a new ring structure. Forexample, an enryme that catalyzed this reaction would be a Iyase:
ATp *.4p1p + pp;
Lyases differ from other enzymes in that they only require one substrate for the reaction inone direction, but two substrates for the reverse reaction.
An exampl. of ty.r. is fumarase. tricarboxylic acid cycle enzyme. lt catalyzcs the reversibleconversion of fumarate to malate. ln this reaction occurs or disappears double bond
o\ /o'?c-HilH_?
{c-o-f umarate
+ HzOfumarase
o\ /o'c
IHO-C-HIH-C-HI{c-o'
malate
4T
beto
ISOMERASES
lsomerases catalysze the isomerizationpfioshofriose isomerase, It conveilsdihydroxyacetone PhosPhate. .
reactioJts. An example of isomerase mayreversibly glyceraldehyde 3'phosphate
o\ /HG
I
H_C-OHI
phoqhotriose
HI
H_G_OHIG:OH-f-o-OI
H
dihydroxyacetone Phoshate
H-C-OI
H
,sornerase
glyceraldehyde 3-PhosPhate
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SYNTHASES oT LIGASESSynthases are enzymes that catalpe synthesis reactions at the expense of energy derivedfrom ATP (or other triphosphate nucleotide). For example, enryme glutamine synfhehse isbonding of ammonia with y -carborylic group of glutamate, forming a glutamine. ln thisreaction one molecule of ATP is used.
ooa,,,o-
I
CHzl-?,,H-C-NHs*
I
o'c-o-
glutamate
glutamine synrherase
Oo"-NHzI
?,,CHzl-H-q-NHs*
I
o'c-o-
glutamine
tq
The mechanism of biocatalysis on the example of selected enzymes
The mechanism of biocatalysis has been fairly well understood for a number of hydrolyticenrymes, i.e..lysozyBe,
.carborypeptidase A
.chymotrypsin.
Lysoryme is a protein enzyme with a relatively simple structure: it contains 129amino acid residues. There is no prosthetic group. lts spatial structure is stabilized by fourintramolecular disulfide bridges. Substrate of lysozyme is a polysaccharide of the bacterialuall, built from repeated manytimes dimer a complex of N-acetyl muramic acid and N-acetylglucosamine. Both these oomponents are bonded by 1,4 -p-glycosidic bond. Thisbinding is degraded by lysozyme.
On the surface of lysoryme molecules is a polysaccharide binding cavlty. Crucialtobiocatalpis have two carboryl groups.These are:
.non-ionised carboryl group of yglutamate at position 35
.and the ionized carboryl group of p -aspartate at position 52.
B
2t
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Carboxypeptidase A
carboxypeptidase A is a proteolytic enzyme, it separates from the protein (peptide)' asingle i+eiminat amino acids, exceS tysine and arginine. lt has a particularly high activity.g.]*t peptide bonds formed wiflr the participation of amino acids containing aromaticring or long chain. Carboxypeptidase A is a protein single'stranded, built of 307 amino acid
residues and 3g percent of them have an a-helix structure. Enzyme molecule is compact,
has a etipsoic shape, and the active site contains a covalently bound zinc ion (Zn2*). Jon,This ion produces a coordination bond with two histidine side chains, side chain ofglutamate and a water molecule. Thanks these connections, the water molecule becomes
iery rea*ive. Bonding of substrate by enzyme causes significant changes in the activesite of the enzyme.
CarboxypePtidase ACarboxypeptidase A usually refersto the pancreatic exoPePtidasewhich hydrolYzes PePtide bonds ofG-terminal residues with aromaticor aliphatic side chains.
CPA-I and CPA'2 (and PresumablYall other CPAs) contain a zinc atomat the active site. Loss of the zincleads to loss of activitY, which canbe replaced easilY bY zinc, and alsoby some other divalent metals(cobalt, nickel).
Z, I
#-f
.d
rg
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LysozymeIt is also known as muramidase or IV-acetylmu ramide glycanhydrolase, a reglycoside hydrolases, enzymes thatdamage bacterial cell walls bycatalyzing hydrolysis of 1,4-beta-linkages between N-acetyl muramicacid and N-acetyl-D-glucosamineresidues in a peptidoglycan andbetween N-acetyl-D-glucosamineresidues in chitodextrins. Lyiozymeis abundant in a number ofsecretions, such as tears, saliva,human rnilk, and mucus. Jt is alsopresent in cytoplasmic granules ofthe polymorphon uclear neutrophils(PMN). Large amounts of lysozymecan be found in egg white.
NAG = N-acctyloglucosamine
Reaction catalyzed by lysozyrneNAG NAM NAG
Z3
NAM
NAM = N-acotylmuramrc actd
NHI
f:oCHs
k\.{\l
NAG NAIVI
2q
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Chymotrypsin
Chymotrypsin is also a proteolytic enzyme. Unlike carboxypeptidase does not cutthe C-terminal amino acids only hydrolpes peptide bonds located in the depths of theprotein chain, which teads to fragmentation of the substrate to peptides of various lengths.Chymotrypsin is a protein composed of three polypeptide chains, joined by disulfide bridges.Chymotrypsin active site: serine 195, histidine 57 and aspartate 102.
Chymotrypsin preferentially cleaves peptide amidebonds vyhere the carboryl side of the amide bond (the P,position) is a tyrosine, tryptophan, or phenylalanineThese amino acids contain an aromatic ring in their sidechain that fits into a'hydrophobic pocket' (the S,position) of the enzyme. The hydrophobic and shapecomplementarity betnueen the peptide substrate P,sidechain and the enryme S, binding cavrty accounts forthe substrate specificity of this enzyme. Chymotrypsinalso hydrolyzes other amide bonds in peptides at slowerrates, particularly those containing leucine at the P,position. L)-
Conversion of glucose-6.phosphate, depending on the enzymeDepending on enzyme used the reaction can run in different directions. For example,glucose-$-phoshate can be transformed into different products depending on the enzyme.
H
OH H
o-'w:'slucose-lO eo
I
CH
OHH
rructos@
DHG-6-c
H
s-C-glucolactone
PGM = phosphoglucomutasePHI = phosphohexoisomerase
@ = phoshate
DHG-6@ = dehydrogenase glucos"'6-@G-6-F = glucoSe-E-phoshatase
z6
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AI losteric regulationln biochemistry allosteric
regulation is the regulation of anenryrne or other protein by binding anetfector molecule at the protein'sallosteric site (that is, a site other thanthe protein's active site). Effectors thatenhance the protein's activity arereferred to as allos teric activafors,whereas those that decrease theprotein's activity are called allostericinhihitors. The term allostery comesfrom the Greek allos "other", andsfereos - "solid (objed),,
Usually, effectors arethe small molecule of metabolites, which by combining withenzymes change their tertiary and/or quaternary structure. Their presence alters thereac,tion kinetics, which is manifested in changes in the Michaelis-Menten's graph.
The allosteric activator makes that the maximum of reaction rate (Vrj isachieved at a lower pH. As a result, the Ku value decreases.
The allosteric inhibitor causes the opposite effect A value of K, increases.
Goenzymes necessary during biotransformation
COENZYTVTE KIND OFREACTIONNAD*/NADH Addition/elimination of hydrogen
NADP+ / NA As aboveATP PhosphorylationSAM Cr -alkilation
Acetyl - CoA C2 -alkilationFlavins oxidation
Pyridoksal phosphate transaminationBiotin carboxylation
metalporphirincomplexes
a) other triphosphates such as: GTR crP and UTP are working the similarly way.G- guanidine; C - citosine; U - uracil.
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oxidationperoxidation
ti
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Coenzymes can be classified depending the group, which transferthey facilitate
Ilydrogen transfering, H, coenzymes:
NAD*, NADP+FMN, FAI)Lipoic acidCoenzyme Q, CoQ
Coenzymes transfering other groups than HCoA-SHThiamine pyrophosPhatePirydoxal phosPhateFolate coenzymesBiotin ? {/Cobamide coenzyme (coenzYme Brr) L I
Nicotinamidadenine dinucleotide' NAD*Nicotinamide adenine dinucleotide, abbreviated NAD+, is a coenzyme
found in all living cells. The compound is a dinucleotide, since it consists of twonucleotides ioined through their phosphate groups, with one nucleotidecontaining an adenine base and the other containing nicotinamide.
nicotinamide
fi ade uineN
ribose#
rihose
N
OH
o
Phosptrate addition G esterrs bond\-/( in place shon'n [' arrou') nicotinamidadenine phosphate (NADP+) is fornred
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pantethenoic acid
Coenzyme A
ffio-o-f*
|
trIoI
o
cystamine
#
HS.. ,CH',,CHz I:{
I
H
f". -Q-CH2-O-\/f' 'cH'OH
The components ofCoA and- SH groupare indicated .Thiolgroup is the bindingsite of the acylresidues
oAI cHrI'-'cI/, -'T'
H
#p-alanine OH
+pante na te
Pantothenic acid is the amide formed bythe bond between pantoate and p-alanine.
Pantothenic acid is used in the synthesis of coenryme A (CoA). CoA may ac't as an acyl groupcarrier to form acetyl-CoA and other related compounds; this is a rvay to transport carbonatoms within the cell. CoA is important in energy metabolism for pyruvate to enter thetricarborylic acid cycle (tCA cycle) as acetyl-CoA, and for o-ketoglutarate to be transformedto succinyl-CoA in the cycle. CoA is also important in the biosynthesis of many importantcompounds such as fatty acids, cholesbrol, and acetylcholine. it
Coenzyme A (CoA)
It is a carrier of acyl groups (acid residues). CoA structure can be divided intothree components.
-Diphosphate nucteotide - adenosine diphosphate (ADP)
-. Pantothenic acid (vitamin BJ - a combination of p-alanine and the pantenoic acid
Gysteamine-SH. lt contains a thiol (-SH) group that generates thioester bond with variousoryanic acids. ln this way acyl CoA derivatives are formed, known in short as the S-acyl -GoA This symbol of CoA derivatives is intended to indicate that the binding site of theacyl group is a sulfur atom, and the connection between sulfur-CoA and acyl group(shown wavy line ,,- ,,) is a bond rich in energy, ln this pathway the acid residues areactivated. ln this form they may be substrates for the synthesis of various esters such asacetylcholine, acylglycerols, or turned to catabolic pathways such as: p-oxidation of fattyacids or tricarboxylic acid cycle.
3>
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Lipoate/lipoamideLipoic acid (l-A), also known as o-lipoic acid is an organosulfur compound derived from
octanoic acid. l-A contains two vicinal sulfur atoms (at C5 and C8)attached via a disulfide bond andis thus considered to be oxidized. This thioacid occursi in two forms. The reduced form has two-SHgroups at the C6 and C8 (dihydrolipoate), and the oxidized form contains a disulfite bridge (lipoate).Carboxylic -COO group is combined with the enryme through an amide bond of e- NH2 of Lysresidues, therefore a coenzyme is called lipoamide. Lipoate is an intermediate acceptor of acylgroups in the process of oxidative decarboxylation of a.amino acids.
Q Hz- CHz-QH-CHz-CH z-CHz-CHz-{-o
lipoate
Flavinmononucleotide (FMN) and Flawinadenine dinucleotide (FAD)
dime thylisoalloxasine dime thyisoalloxasine
l atlenine
FMN ] riboseFAD
Flavin adenine dinucleotide (FAD) is a redox cofactor involved in several important reactionsin metabolism FAD can exist in two different redox states, which it converts between byaccepting or donating electrons. The molecule consists of a riboflavin moiety (vitamin Br)bound to the phosphate group of an ADP molecule. FAD can be reduced to FADHI whereby itaccepb two hydrogen atoms (a net gain of two electrons):
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S-AdenosYl methionine (SAM)S-Adenosyl methionine (SAM, SAMe, SAM-e)is a common co-substrate involved in methylgroup transfers. the meihyl group (CHr) attached to the methionine sulfur atom in SAM isihemically reactive. This altows donatiLn of this group to an acceptor substrate intransmethylation reactions. More than 40 metaboiic reactions involve the transfer of a methylgroup from SAM to various substrates such as nucleic acids, proteins, and lipids.
methionine
SAM
Thiamine pyrophosphate (TPPThiamine pyrophosphate (TPP, derivative thiamine, vihmin Br) is a coenzyme that is
present in al! living systems, in which il catalyzes several biochemical reactions. lt uas firstdiscovered as an essential nutrient (vitamin) in humans through its link with the peripheralneruous system disease Beriberi, which resulb- from a deficiency of thiamine in the diet.
TPp participates in the process of oxidative decarboxylation of o-ketoacids (pyruvate, a'ke{oglutarate}. Thiamine binds a -ketoacid through its carbonylgroup. Next, the elimination ofCO2 occurs and reaction's carbonyl produc't is transfered to followig acceptor.
aldehyde binding site 1,
Nl-.b
''
/
TL -@
CII
-5)
Thiamine pyrophosPhate
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Pyridoxal phosphate, PLPPyridoxal-phosphate (PLP) is a prosthetic group of some enzymes. lt is the active
form of vitamin Br called pirydoxamine. PLP acts as a coenzyme in all iransarninationreactions, and in some decarboxylation and deamination reac'tions of amino acids.
oo"-H
aor\
\.jA.\JO(r\c
Hz -c H z- cHz-cHz -
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Keactron catalyzed by bfunctlonal enzymeBifunctional enzymes generally contain two large structural domains whoseassociation facilitates metabolic pathway control and/or allows more efficientsubstrate conversion.
W;I_I'OHHfructose-6-PhosPhate
ICHzOH
t
fnrc tose -2,6-bis- Phos Pha te
Phosphofructokinase 2 (PFK2) is angluconeogenesis in the human body.
6-G)phos phofnrctokinase -2
fructose -2,6- bis- phos pha te
fructo kinase -2,6-bis- phos phot aseOHH
fmctose-6-phosphate
@ : plrosphate
enzyme responsible for regulating the rates of glycolysis and
tCHzOH @' Y'UI*ow,,[-o
rl
Kinetics of enzymatic reactions
@o-.l
4
ho
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Thermodynamic effect of reactionThe exergonic pr6r.ss is therrnodinamically favorabte.
A (suUstrate) --+ B lProOuct)
Progress of reaction
Exergonic process
C (suUstrate) ---+ D 6toduct
G
Start
Progress of reactionEndegonic process qt
G
Effect of enzyme on the value of free energy, G,
+- withoutenzyme
G
Progress of reaction
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FORMAL KINETICS OF REACTIONS CATALYZED BY ENZYMES (1)
E + s -k-t 'k-r ES
k*'- products
v(rate): k*2 I ES I 1.2liwe denote the equilibriurn constant I as the dissociation constant of the
complex ES to E (enryme) and S (substrate), and if the [E'l denotes the initialconcentration of enzyme, then the concentration of free enzyme is ([E'l - [ES]. Then:
From equation 1.2 we get: v _ tEl_lql: (tEI-tESI)tsl r.3^'=[Eq - rest
0Eltsl - [Es] [s])
1.1
K"=
v:
[ES]
k.,[E]1+ K'
tsl
1.5
4s
FORMAL KINETICS OF REACTIONS CATALYZED BY ENZYMES (2)
When the substrate concentration S is very high, then the equation (1.5) determinesthe maximum rate of reaction V and is close to the expression
V = k*z [E']
By inserting this expression for V in equation 1. 5 we obtain:
v: 1.6Kl+ stsl
This is so called the Michaelis - Menten's equation
Michaelis constant, G, is defined as the concentration [Sl, when the reaction reaches half themaximum rate (V). The value of lq can be obtained experimentally from (1'6). Although it ispossible to determine tfi from the slop, but the accuracy of this method is low due to theasymptotic nature of dep-endencie. ln practice, it is prefered to use equation (1.7) obtained byrearranging (1.6):
1L+tS] V
1
Vt.7
hLt
-
:MICHAELIS. MENTEN'S PLOT
tsI
Dependence of reaction rate v on substrate concentration [Sl under constant concentration ofenzyme, [EI = const. At high substrate concentrations the reaction rate (v) reaches its maximumvalue V.
K, is equal to substrate concentration, [sl, when the value of v = 0.5 v. b{
LINEWEAVER I BURK'S PLOT(double reciprocal)
1/lsl
Dependence of lfu from 1/ ISI for enrymaticalty catalyzed reactions described bythe Michaelis'Menten's equation
According to the Michaelis-Menten's kinetics, K, = l(.Therefore, the K, is a directmeasure of binding forces between enzyme and substrate.
The above plot is also very useful to study the inhibltion of enzymatic reactions.
sbclsse _ -l I Km
-
lnhibition of enzYmeslnhibition is the phenomenon of decreasing of enzyme activity.
Substances that inhibits the enzymes are called the inhibitors. lnhibitors are divided into twogroups: competitive and non-competitive.
The competetive inhibitor is structurally similar to the substrate. lt binds to the enryme in itsactive site causes decreasing of affinity of substrate to enryme and increasing of K, .lnhibition is reversible by increasing the concentration of the substrate. Vro does notchange, but it is achieved with higher concentrations of substrate. An example of thiskind of inhibition is lowering activity of enryme succinate dehydrogenase,whichoxidizes succinate to fumarate with the participation of FAD (Flavin AdenineDinucleotide). lnhibitors with a similar structure are: malate, oxaloacetiate and oxalate).
The non+ompetitive inhibitor is not similarto the substrate. lt binds to the enzyme outsidethe active site. Distorb the enryme protein molecule, and thus reduces its activity.Thiskind of inhibitor does not alter the affinlty of enzyme to substrate, also K, does notchange. lt reduces Vmar. The inhibition can not be reversed by increasing substrateconcentration.
Michaelis-Menten (A) and Lineweaver -
Burk's (B) plots for thereaction with a competitive inhibitor
(+) inhibiton
(-)inhibitor
lff*"*: lff*r-(i)
-llI(M -lfifu(i)
t4T
K*(DB
{,t C
inhibitor
(+)inhibitor
KM
-D* ^ - L'4,
tsI
-
vMichaelis-Menten (A) and Lineweaver -
Burk's (B) plots for thereaction with a non-competitive inhibitor
IN(+) inhibitor
(-) inhibitor
Krra:Knr(i) ISI lfffu:llKM(i)
Differences between competetive and non-competetive inhibitors
vrr*
V*"*(i
Vr"*/z
Vrr(i)2
tSI
B
h\A
lN ^r_(i)
(-) inhibitor
(+) inhibitor
Competitive inhibitor Non -cornpetitive i n h ibitor
Structure Similar to the substrate Non'similar to the substrate
Bsnding site Aetive site Outside the active site
Reversibility lnhibition is reversibledue to increasing ofsu bstrate concentration
Reversible inhibition by anincrease in substrateconcentration
V*.* No change Decreasing
KM lncreasing No change
-
Oxidation of succinate by succinate dehydrogenaseand competitive inhibitors of this enzyme
A = OXIDATION OF SUCCINATE
FAD FADHZoo-\/c
IH_C-HI
H_C_HI
ooc'o'
succinate
oo'\/c
IH-Cilc-HI
o'c'o'
fumarate
oo"-o'
IH-C-HI
o'c-o'
malate
oENZ-SH + I_GHZ-8-NN'
oENrc-cHz-c-Un,
FAD - Flavin Adenine Dinucleotide
B = INHIBITORS OF SUCCINATE DEHYDROGENASE
Non+ompetitive inhibitors and their mechanisms of action
HIJ
oo'oc'I
o'c-o'
oxalate
oo'oc'I
H_C_HIc:oI
o'c'o'
oxaloacetate
s{z-o,*"
, Ght2cH2
-OG-C+'lz4 rrcH2-COO'CIF*2 Ct{e
OF-O--O-C=O
qH(cH3)2I
oIENZ-o-P:oI
oI
CH(CH3)2
,CH2'G$2-ooc-cHz-T 'ry-cH2-GOo'
?H, ?H,o"'o o'c=o\/
Mg
Mg'*(,
Bonding of enzyme -SH groups by iodoacetamid
?H(cH3)2o
IF-P:OI
?cH(cHg)z
HFJ=cnCrg d -3r- 3ra.e C serire reskJues in enzyme by diisopropylofluorophosphate
Bordrrg cf }tq-no{ns Q przz dfryErderninetetraacetate (EDTA} fL
-
Lineweaver =
Burk's PlotDependence of 1/V vs 1/S for the enzyme with different substrate affinity
(high affinity) and B (low affinitY)
lnfluence of allosteric effectors on reaction rate (V) versus substrate. concentration [S]
vrr*
K** KM h*r,
- allosteric activator
- allosteric inhibitor
A
-j)
o
o
1/KM(A) 1/KM(B) 1/[sl
-
Dependence of enzymatic reaction rate (V) on temperature
ln the temperature range 0'40oGdoubles the speed of reactioJl.enzymatical reaction due to theabove 60oC.
l0 20 30 40Temperature l'Cl
reaction rate increases. The increse of temperature of 1()oCThe increase in temperature >40oC reduces the rate ofdenaturation of the enzyme. Sorne enzymes show activity
J-r
Dependence of reaction rate catalyzed by certain enzymes on pH
Various enzymes have different pH optima. Some enzymes are not sensitive to changes inpH. For example, papiin is equally active in the pH range 4 '8.
\'
i6
-
Michaelis-Menten's plot showing the dependence of V (S) for theenzyme with different affinity for substrates A and B
vrr* {----
Enzyme A has a high aflinity for substrate
Enzyme B has low affinity to substrate
Kurnl Krtul ISI
Enryme reaction rate, V increases with increasing substrate concentration, S. ln a somerange of concentrations the reaction rate is linearly dependent on subotrate concentration.The dependence curue of V on S has a hyperbolic shape. Wth sufficient substrateconcentration reaction rate reaches its maximum value, Vro.
Michaelis-Menten plots of dependence of V on [S] glucosefor hexokinase (H) i glucokinase (G)
Vru*(HY2
fl
Kr(H) Kr,a(G)
hexokinase
glucokinase
glucose
v*u*(Gy2
-
Enzyme Substrate KM [mollt,lFumarase fumarate 5r0 x 10-6
Penicillinase benrylpenicylline 5r0 x 10-s
Acetylchol i ne esterase acetylcholine 9,5 x 10-s
F-Galactosidase lactose 4r0 x l0-3
Carbonic anhydrase (orcarbon ate dehyd ratase)
co, lr2 x 10-2
Urease urea 2r5 x l0-2
Examples of values, of Michaelis constant
Maximum value of the speed of some enzymes
f{
[L,,,w;rii,,Srit:i
Enzyme Number of molecules ofsubstrate pes I sec
Carbonate anhydrase 600 000Acetylchinoline esterase 25 000Phosphatetriose isome rase 4 400
I-actate dehyd rogenase 1 000
iEfmotrztpsin&
100
11
ffinsynthetasek$ 20r5
5,a
-
Pharmacological use of enryme inhibitorsEnzyme lnhibitor Etfect Application
Acetylcholineesterase
prostigmin Increase of concentration ofacetylcholine trigger arnuscular contraction.
Drug triggering thecontraction ofintestines
Monoamino-oxyda$e
selegiline lncrease of dopamineconcentration in brain.
Used in newlydiagnosed patientswith Parkinson'sdisease
Phosphodiesterase Theophylline I ncreasi ng of concentrationof cyclic 3',5',-AMP in cel!
Used to treatasthma andchronic obstructivebronchopulmonarydisease.
{t
Pharmacological use of enzryme inhibitors (cont.)E,nzyme Inhibitor Effect Application
Carbonateanhydrase
acetazolamide lncreased secretion ofbicarbonate andphosphate by the kidney
Diuretic
Xanthineoxydase
allopurynol Reducing the productionof uric acid
Drug for gout
BloodEeagulatiqnfactors: IXaand Xa
citrate Binding of Caz* ions lnhibition of bloodseagulatipndestined fortransfusion
Cyclogenase Acetylsalicilicacid
lnhibition of synthesis ofthromboxanes andprostaglandins
Anti-inflammatoryand anti-thrombotic drug.Preventingclotting
67-