2eN6EsU)

\

a!h,II.J

-cII\(1)

ENF-o,F-(tro

t|i,tTc,(I,F-

(J(tr{r,{E,ctr

-b{r,e--g(uo(J

{JoFr-o,l-G'aL-ottr,(J(\,

t{ro(J

tfr,aoE.R .lC,L- 1;or=

\ Jg^ge

-=)tf {r,E ?Its,li .9?=

{r'

>\ l-NG'tE()rtr

(triog-ftt(J.9

I

o,ENer-I

o,ocl.(trFft{r,'=

.>{r,F-IG'o(JIF-oFr-rEo,

{r,(\tl- (Jooo(U

.c2tEo

tfr,(J(trf{ro(,(l,?

I-

*r,

?-o,?i

=

T'q,-

(U(J.(2.E

Io,ENF-(1,oCLfiTo

*r,

->{ra?-q,(tro(Jt,Fr-

rIao-cI.9L-o{r,fJ(trqro(JFr-'6{r,oL-CLIF-oe-o,?

-

{r,Fr-o,Ffr

=

o,ENFIE,oa-(U+

II(1,ENEo,qo

-

J.

atEN(r.r-,ooFr-

'lc,qII (tr(J.r2(I,tr-

N?r-,oCLG'

F-c,c,

=!h,G'

-cIFr-orI{r,(Jo,gFr-o()(u?ft{r,

'lc,g(G

h6'ENF-o,ocl-(trE(1)I (U(J.9,(1,ENFr-r(I,

(trEo)-

(tr(Jh

tlr,erLo,F-()cl-Eo(JF-'6

tfJoL-cl.IFr-oF-EFr-(trFr-'6!tr,oL-CL(U

ra-oo{r,.93n(--o(J

x(l,CLEofJq,ENE(1,o,e-F

Ixo,CLEo(,

'tc,E(tro,CL.Ea{r,(uF

I-{r,oer-'6{rtoL.a.(l,I-(tra,

ENF.-

ut

He-;L-(trCLct,er-l

=(tr

tfr,

oo,tfr,G'L-tar,a

-clI-Joa(tr

haFr-ro

II

!fr,()Ctr,L-()

III

{r,

->c,{-,c,(,

rfrooELo,tH(r--

t;

.I-{r,o-cI>i=(JIIl{r'6q,CLoFD.9eI-

-cI:c,(uht

II L-(I,ttr,(J(\,L-IUer-(Jo,t-a,oo,ENFi-

UII

o,

Egcr)o,r--I

.9,ttr,(Jo,r+rq-o,a,=!+r,(trFft

{r,

Eoa

PsH'A as,t o' E(, *.9 2crrc.gij=

gEE q =FfrEgaEggEE

;IG= (trE'rd E 3E=.8

B g o.C-CI-t ,* ,oS:g3EE Eft-EE gHE FET Eid or= 5

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 ).

3

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

F'f',:r,-lLt,- t: I t rll"'i lill,-1.r::flr. ::.it*,:,f rfl l."t-ft*

'I'. Lt Lr :;['at-r a n[et'l n!la,lti"i* rit-i ,.,f rl]r','lJ'rr

E n :'r'ttr *i .: r-t li 5 tr';rlr'-:

r.r l'l'1pi | ; .1

,.1.,] tii.rEE ite

pJr

ir= tii'e: ttr=

5;r.t

E tt .;r'ttl tr/ [' 1'';r r-ll-t'-:t-l;r- r-rl-;-spl*;f

lnduced fit model

(a) Reacti sltSt-rbet'4.t3

Et r:ynrH"'

{h} Iltltihitiotlh rlr ib it':l'

tt'*

{

\

B

ffiffiP-it

Err =ylrre h irr':l: sultst'ate Etr :;,nIB r r lraae: 1rr-'rrlt-r*rs

lrflf il.' it:t' ,::,--iptt:rtsIE5',t itr i-Lrhstr'e.r*

Etr rytllE *

Err :;,rrre h irr:l:-' inlr ih in-'r'

-$ubstrate

EnzyrneEnzyrne -SubstrateComplex

lnduced-lit Modet. - The enzyme active site forms a complemehtaryshape to the strbstah after binding,

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

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

_n\,,

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.

IL

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'

/t

l,'"8

.,'',u'

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,

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

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

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

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

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

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.

74

oxidationperoxidation

ti

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

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>

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):

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

36

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 -

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

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

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-