Chng V- ENZYME

BI GI NG SINH HA H C
PH N I SINH HA H C TNH Chng V- ENZYME

1. 2. 3. 4.

Enzyme v hi n t ng xc tc sinh h c C ut o C ch ho t ng xc tc Cc y u t nh h ng n ho t l c xc tc c a enzyme 5. Danh php v phn lo i

TP.HCM-2008

M C TIU
1. Ban chat hoa hoc cua enzyme? Nhng ac iem cau truc chung cua enzyme, trung tam hoat ong cua enzyme. 2. Giai thch c che xuc tac chung cua enzyme. 3. Tnh ac hieu cua enzyme va cac ieu kien moi trng anh hng en kha nang xuc tac cua enzyme ? 4. Danh phap va phan loai enzyme theo 6 lp. Thanh phan cau tao va hoat ong xuc tac cua moi lp.

1. ENZYME V HI N T NG XC TC SINH H C Enzyme l nh ng protein gi ch c nng xc tc cc ph n ng sinh ha h c. Nh E xc tc m cc p SHH c n thi t cho s sinh s n c a t bo x y ra v i .t c . c tnh cao, c hi u cao, s ng v s

. ti t ki m NL. E c th xc tc
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i u ki n in vivo v in vitro.
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2. C U TAO C A ENZYME Phn t enzyme l cc protein d ng c u, ty lo i E, chng ho t ng d ng c u trc b c ba ho c b c b n. E m t c u t protein g : TP bao g m cc AA, ch 1 chu i ho c 2 hay nhi u chu i polypeptide gi ng nhau hay khc nhau. E nh c u t - protein ph c t p : Protein /g + (apoenzyme) K t h p c hi u v i c ch t
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Xc tc l hi n t ng lm . tng t c b ng ph n ng, t t i tr ng thi cn p g i l ch t xc . h th ng p chng Ch t lm tng t c

ng b ng nh ng ch t a t ngoi vo.

tc, chng ch tham gia trong cc SP trung gian c a p , chng khng c m t trong s p cu i cng c a p .

nhm ghp (cofactor) (coenzyme) Tr c ti p xc tc ph n ng

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Tr ng h p apoenzyme lin k t ch t ch v i coenzyme CoE c g i l nhm ngo i prosthetic group. Tr ng h p apoenzyme lin k t l ng l o v i coenzyme, khi ho t ng xc tc CoE tch kh i apoE CoE c g i l cosubstrate

2.2. TRUNG TM HO T

NG C A ENZYME

L vng ti p xc c a E v i c ch t (S = substrate) trn phn t E, l ni x y ra qa trnh xc tc. TTH hnh thnh do s s p x p c a m t s AA chuyn bi t, n m xa nhau trn polypeptide nhng g n nhau trong c u trc khng gian : - Cysteine SH - Serine - OH
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N NH
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- Histidine

TTH c 2 ac iem quan trong : hnh d ng TTH ph h p v i hnh d ng phn t c chat m n xc tc; i n tch TTH tng ng nhng tri d u v i i n tch phn t c chat. Fischer's lock and key hypothesis of enzyme action.

Cc i u ki n mi tr ng (nhi t , pH ) nh h ng n c u trc b c ba c a phn t protein enzyme u nh h ng t i hnh d ng v in tch c a TTH nh h ng n ho t l c xc tc c a enzyme.

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Diagrams to show Koshland's induced fit hypothesis of enzyme action.

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3. C CH HO T

NG XC TC H2O2 H2O2 H2O2

khng xc tc b t platin catalase

Thuy t h p ph : ch t xc tc h p ph c ch t ln b m t chng t p trung S, tng n ng S tng v n t c p/ (h th ng p/ d th : ch t xc tc th r n, S th l ng, kh). Thuy t l p h p ch t trung gian : ch t xc tc t o h p ch t trung gian v i S, p/ i theo ng vng gi m NL i h i ho t ha c ch t tng v n t c p/ (h th ng p/ ng th : ch t xc tc & S cng th l ng, kh). 13

H2O + O2, NLHH : 75 kj/mol H2O + O2, NLHH : 49 kj/mol H2O + O2, NLHH : 8 kj/mol

E l protein v a c k/n h p ph cao, v a c k/n TL h/c trung gian E c k/n xc tc cao hn nhi u so v i ch t v c.

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4. CC Y U T NH H NG NV NT C PH N NG ENZYME n v ho t lc c a enzyme Bi u th tr c ti p : kh i l ng t s d ng v kh i l ng enzyme th ng r t th p so v i kh i l ng c ch t m n xc tc. Bi u th gin ti p : ho t nhi u. c s d ng

Nng l ng ho t ha c n cung c p cho m t ph n ng

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n v qu c t ho t c a enzyme IU (international unit) : l l
ng enzyme xc tc bi n i 1 mmol c ch t hay t o ra 1 mmol s/p trong th i gian 1 pht d
i i u ki n chu n c a 16 nhi t , pH v n ng c ch t (IU/lit)

H S CHUY N ENZYME SANG

IM TS N V HO T L C N V H TH NG QU C T n v qu c t U/lit U/lit U/lit

M i phng th nghi m th ng bi u th n v ho t l c enzyme theo p/p xt nghi m ring c a mnh so snh ph i i ra n v chu n qu c t SI (International System of Units) b ng cch nhn v i h s chuy n i.

Cc enzyme huy t thanh Aldolase (ALD) Amylase (AMYL) Glutamic oxaloacetic transaminase (SGOT, AST)

n v H s khng h th ng ch/ i Sibley-Lehninger unit 0.75 (SLU)-mgDNP/hour/ml Somogyi unit (SU) 1.85 mg G/30 pht Sigma-Frankel unit 0.48 (SFU); Kamen unit (KU) Wroblewski-LaDue unit (WLU); ReitmanFrankel unit (RFU) 0.001 OD/pht/ml

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Glutamic pyruvic transaminase

U/lit

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Glutamic pyruvic transaminase (SGPT, ALT) Isocitrate dehydrogenas e (ICD) Lipase Phosphatase acid (AcP) Phosphatase ki m (AlP)

Sigma-Frankel unit (SFU); 0.48 Kamen unit (KU); WroblewskiLaDue unit (WLU); ReitmanFrankel unit (RFU) 0.001 OD/pht/ml Volfson-Williams-Ashman unit 0.0167 (WWAU) nmol/gi /ml Roe-Byler unit (RBU) ( mol/gi /ml) King Amstrong unit (KAU) mg phenol P/30pht King Amstrong unit (KAU) mg phenol P/30pht Bodansky unit (BU) - mg P/gi 1.85 7.10 5.40 16.7

U/lit

Ghi ch : U = 1 international unit = 1mol/pht = 16.67 nkat/giy = 0.0167 kat/giy

U/lit

U/lit U/lit U/lit

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4.2. NH H NG C A NHI T 4.1. V N T C BAN U C A PH N NG i u ki n in vitro : 0 400C : to tng t c p/ tng, n thu n nhi t c/c NL cho p . >450 C: v n t c p/ b t u gi m do nhi t lm bi n tnh protein. >800 E b bi n tnh hon ton. . E ngu n g c /v : t0 t i u : 40 500C . E ngu n g c t/v : t0 t i u : 50 600C M t s VSV ch u nhi t, s ng trong su i n c nng 70 800C. i u ki n in vivo : nhi t c th tng 1-20C p/ t chy ch t h u c trong m bo tng hng trm l n gy s t, l p/ b o v c a c th . 22

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4.3. NH H NG C A pH pH mi tr ng thay i nh h ng n s phn ly c a cc nhm ch c trn chu i polypeptide thay i i n tch TTH a/h kh nng k t h p E + S gi m v p/. M i E ch ho t ng t t trong m t gi i h n pH nh t nh, ngoi kho ng pH t i u th v n t c p/ r t th p. B ng 5.2 (T.112) : kho ng pH t i u c a m t s enzyme.
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pH t i u c a m t s enzyme Enzyme -Amylase (n c b t) Pepsin (d dy) Trypsin (t y t ng) Lipase (t y t ng) pH Enzyme pH

6,8 7,0 -Amylase 5,2 (m ch nha) 1,5 2,5 Cathepsin 4,5 5,0 (c) 7,8 9,5 Phosphatase 9,0 - 10 (huy t tng) 7,0 8,0 Sucrase (n m men) 4,6 5,0
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4.4. NH H NG C A N NG V n t c p/ tng t l thu n v i s enzyme trong h th ng p/

ENZYME tng n ng

4.5. NH H NG C A N NG C CH T N ng S tng v p/ tng, khi t t c cc TTH c a E u t/g xc tc v p/ t t i a.

Vn tc ban u t l thun vi nng E

Lng c cht bin i

3x 2x 1x

E3 E2 E1

t0

t1

t2

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4.6. S

HO T HA V

C CH ENZYME

HO T HA ENZYME Trong m bo E khi m i c ti t ra th ng tr ng thi cha ho t ng (ti n E, ti p u ng pre hay proenzyme, ti p v ng ogen.

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Cat bo mot oan polypeptide cua tien enzyme (proenzyme hay zymogen) lam boc lo trung tam hoat ong. V du : Pepsin + HCl pepsinogen pepsin + peptide (42.000) (35.000) (7.000) Thanh lap cau noi disulfur (-S S-) e hoan chnh trung tam hoat ong : Enterokinase Trypsin + heptapeptide30 Trypsinogen

Thanh lap phc hp vi cac ion kim loai, cac ion nay co vai tro lam cau noi trung gian, gan c chat len trung tam hoat ong bang cac cau noi phu. Vai tr ny th ng thu c v cc ng/t vi l ng. Hoat hoa nh hien tng cam ng b i chat gay hieu ng d khong gian. S ho t ha (hay c ch ) E th ng c ki m sot b i s phosphoryl ha hay s kh phosphoryl : E khng H
HH b ng phosphoryl ha b i kinase + ATP
c ch b ng kh phosphoryl b i phosphatase H3PO4

C CH ENZYME a s cc tr ng h p ng c l do c ch ho t ng c a trung tm ho t ng c a enzyme. Th d : (-CN) lin k t v i Fe3+ c a cytochrome oxidase c ch chu i h h p m bo. c ch c nh tranh (H.5.8, T.119) : (-CHI COOH) c nh tranh v i g c (- CH2 SH) c a Cys trong TTH . c ch khng c nh tranh : ch t c ch lm bi n i c u hnh phn t E lm thay i kh nng xc tc c a E.

E ho t

ng
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Diagram showing the mechanism of non-competitiv e inhibition.

Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.
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5. DANH PHP V PHN LO I DANH PHP Nhm E tiu ha c g i tn ty ti n Quy t c g i tn : theo tn Latin c a c ch t + ki u ph n ng + ASE C chat Enzyme - Amylum (tinh bot) Amylase - Proteinum (protein) Protease Phan ng Enzyme - Van chuyen goc (-CH3) Methylferase - Thuy phan Hydrolase - Kh n c Dehydratase 35

PHN LO I

6l p Ph l p Ph c a ph l p Enzyme

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Th du : Enzyme mang ch so E.C 2.7.1.1 Ch so 2: E thuoc lp 2 - Transferase, van chuyen cac nhom chc. Ch so 7 : phu lp 7, van chuyen nhom phosphate (-H2PO3). Ch so 1 : phu phu lp 1- chat nhan nhom (H2PO3) la mot alcohol. Ch so 1 : enzyme la hexokinase. Tom lai, enzyme mang ch so E.C 2.7.1.1 la E co ten va chc nang nh sau : D- hexose-6-phosphotransferase, xuc tac phan ng van chuyen nhom (-H2PO3) t ATP sang nhom hydroxyl C6 cua glucose. 37

L p I II III IV V oxydoreductase Transferase Hydrolase Liase Isomerase

Xc tc ph n ng kh hydrogen (oxy ha), hydrogen ha (kh ) V/c cc nhm nguyn t v g c p/t t ch t ny sang ch t khc Th y phn (E m t c u t ) Thnh l p ho c phn gi i cc n i i V/c cc nhm ng/t v g c p/t trong n i b p/t thnh l p cc ng phn Thnh l p cc lin k t c n s d ng cc n i P cao nng 38

VI

Ligase

CC ENZYME TH NG S D NG TRONG CH N ON
Vi t t t ALT (GPT) AP Amyl ARG AST (GOT) ChE CK (DPK) Tn th
ng g i Alanine aminotransferase Alkaline phosphatase H th ng phn lo i 2.6.1.2 3.1.3.1 3.2.1.1 3.5.3.1 2.6.1.1 3.1.1.8 2.7.3.2
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Vi t t t GGT GPx LDH LIP OCT SDH PK TK

Tn th
ng g i
-Glutamyl

H th ng phn lo i 2.3.2.2 1.11.1.9 1.1.1.27 3.1.1.3 2.1.3.3 1.1.1.14 2.7.1.40 2.2.1.1 3.4.21.4
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transferase

Glutathion peroxydase Lactate dehydrogenase Lipase (triacylglycerol lipase) Ornithine carbamoyltransferase Sorbitol dehydrogenase Pyruvate kinase Transketolase Trypsin

-Amylase
Arginase Aspartate aminotransferase Cholinesterase Creatine kinase

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(1) L P OXYDO-REDUCTASE (L p enzyme oxy ha-kh ) Xc tc p/
oxy ha-kh (trao i e- ho c trao + + e-). Cc nhm chnh : iH=H Cc dehydrogenase ch a nhn pyridine (d n xu t t vitamin PP) : v/c 2H (2H+ + 2e-) Cc dehydrogenase ch a nhn flavin (d n xu t t vitamin B2) : v/c 2H (2H+ + 2e-) Catalase : p/h y peroxyde hydro g/p O2 Peroxydase : p/h y peroxyde hydro g/p O H th ng cytochrome : ch v/c e41

Dehydrogenase ch a nhn pyridine : - NAD+ : Nicotinamid Adenine Dinucleotide - NADP+ : Nicotinamid Adenine Dinucleotide Phosphate

Nicotinamid

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O C _ O O P OCH2 O H H O OH OH NH2 N N N
O P OH H N

NH2
_ O

NH2

(H+ + e-) H - CO- NH2

H - CO- NH2

N + H H

OCH2 O H H H

N+

H OH OH NH2 N N

+(2H+ N+ R (e-) (2H+

+ 2e-) + 2e- ) N R

+ H+

P OH

N OCH2 O H H OH OH H

N OCH2 O H H

H OH O HO P O
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Dehydrogenase NAD+

NADH + H+
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OH

NAD+

NADP+

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isoalloxazin
O
O H C CH3 C C C H C N C C C NH

Dehydrogenase ch a nhn flavine: -FMN : Flavin MonoNucleotide -FAD : Flavin Adenine Dinucleotide FMN

CH3 C6 CH3

H C 5

C C

N 10

C C

C 4

10
N CH2

3 NH

CH 3

Flavin

C7 8 C H

9 N
CH2

1 2C O N

HCOH HCOH HCOH CH2

HCOH

D-ribitol

HCOH HCOH CH2 O _ O P O O

O _ O P O O _ O P O CH2 H H OH O H OH N HC N H

NH 2
C C C

A
N

N CH

FMN
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FAD

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(H++ e-)
CH3CH3-

N 10

O C 1 N

NH C=O FMN (FAD)

R H
CH3CH3-

(H++ e-) +2H O C 1 N H NH C=O

He thong cytochrome : la cac chromoprotein vi nhom heme cha Fe3+. Co nhieu loai cytochrome, khac nhau ve the nang oxy hoa kh, chung ch tham gia nhan va chuyen electron (khong nhan proton H+). Phan ng c ban : Cyt-Fe3+ +e- eCyt-Fe2+

-2H

N 10

FMNH2 (FAD.H2)
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Trong chuoi ho hap mo bao thng gap cac cytochrome : b , c , a va a3 (cytochrome oxydase ch chuyen ien t cho oxygen). 48

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(2) L P TRANSFERASE

(Cc enzyme v n chuy n)

Xc tc p/
v n chuy n cc nhm nguyn t hay g c phn t t ch t ny sang ch t khc. Phosphotransferase - v/c H2PO3 Aminotransferase v/c nhm NH2 Sulfurtransferase v/c nhm SO3H Acyltransferase v/c cc acid bo

Heme cua cytochrome

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Aminotransferase (Trans aminase) : V/c nhm amine trong ph n ng chuy n amine t/h amino acid trong m bo ng v t. Coenzyme l pyridoxalphosphate (d n xu t t vitamin B6)
H C=O HOH3CN CH2-NH2 HOH3CN

Acyltransferase ho t ta &v/c cc acid bo Coenzyme c tn l coenzyme acyl ha, d n xu t t panthotenic acid : CoA.SH CoA.SH
Pirophosphate

OH -CH2O P=O OH

OH
Adenosine-3- R-C = O monophosphate
A.pantoic -alanine

-CH2O P=O OH

OSH
Thioethylamine SH
-O-

O C-R

Pyridoxal phosphate

Pyridoxamine phosphate 51

A.panthothenic

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(3) L P HYDROLASE (Enzyme th y phn)

L nh ng enzyme m t c u t , g p ph bi n trong ng tiu ha, nh s tham gia c a n c chng th c hi n ph n ng phn gi i cc ch t h u c ph c t p c a th c n thnh cc n phn c th c th h p thu c. Glycoside hydrolase : th y phn LK glycosidic c a glucid Proteinase v peptidase : th y phn LK peptide c a protein v peptide Esterase : th y phn LK este c a lipid Amidase : th y phn cc amid
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(4) L P LIASE L nh ng enzyme xc tc p/
c t tr c ti p cc lin k t trong phn t khng b ng con
ng th y phn, chng c kh nng tch CO2, H2O, NH3 Decarboxylase (tch CO2) Aldolase : c t C6 2 C3 Dehydratase (tch H2O) Deaminase (tch NH3)
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DECARBOXYLASE Decarboxylase c a -ketoacid c CoE l thiamin pirophosphate (TTP) (dx c a vitamin B1thiamine). Decarboxylase c a -amino acid c CoE l pyridoxalphosphate (dx c a vitamin B6).
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NH 2 C N CH3 C N C CH 2 CH + N

H C S _ O C CH3 O _ C CH CH O P O P O 2 2 O O _

Thiamine pirophosphate (TPP)

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(5) L P ISOMERASE (Enzyme ng phn ha) L cc E xc tc s v n chuy n nhm nguyn t hay g c phn t trong n i b phn t t o ng phn. ra cc ch t Glucose-6-phosphate isomerase : chuy n nhm carbonyl C1 C2 (Glucose Fructose). Phosphoglycerate mutase : chuy n g c (-P) gi a C1 C2 trong m t ph n ng ng phn. Racemase : chuy n nhm (-OH) cc ng n, t o ra ng phn dy D hay L.

Phosphotriose isomerase Phosphoglyceraldehyde Phosphodioxyacetone L-Alanine Alanine racemase D-Alanine

UDP-Glucose UDP-Glucose-1-epimerase UDP-Galactose

Phosphglycerate mutase 3-phosphoglycerate 2- phosphoglycerate

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(6) L P LIGASE Xc tc p/
t/h ch t c s d ng nng l
ng t cc n i phosphate cao nng (P) Aminoacyl-t-RNA synthetase t/l p LK C O trong p/ hoat hoa amino acid cua tien trnh sinh tong hp protein. Glutamine synthetase thanh lap lien ket gia C N trong phan ng tong hp glutamine. Ligase thanh lap lien ket gia C - N trong sinh tong hp protein, ti t h p DNA. Acetyl CoA carboxylase thanh lap l/k t C - C trong tien trnh tong hp acid beo (p/ thanh lap malonyl 59 CoA).

CARBOXYLASE : v i CoE l biotin, cung c p nhm

CO2 trong p/ t ng h p acid bo.
Nguyn t N ho t ng

BIOTIN

Phn t

CO2

NH lysine c a apoenzyme

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