101

Chng 6

Enzyme
Enzyme l protein c kh nng xc tc c hiu cho cc phn ng ha hc. Chng thc y mt phn ng xy ra m khng c mt trong sn phm cui cng. Enzyme c trong nhiu i tng sinh hc nh thc vt, ng vt v mi trng nui cy vi sinh vt.Hin nay ngi ta thu c nhiu loi ch phm enzyme khc nhau v s dng rng ri trong nhiu lnh vc nh y hc , nng nghip, cng nghip

6.1. Bn cht ha hc ca enzyme

Ngoi tr mt nhm nh RNA c tnh xc tc, tt c enzyme u l protein. Tnh cht xc tc ph thuc vo cu to ca protein. Nu mt enzyme b bin tnh hay phn tch thnh nhng tiu n v th hot tnh xc tc thng b mt i, tng t khi bn thn protein enzyme b phn ct thnh nhng amino acid. V vy cu trc bc 1, 2, 3, 4 ca protein enzyme l cn thit cho hot tnh xc tc ca chng. Enzyme, cng nh nhng protein khc, c trng lng phn t khong 12.000 n hn 1000.000.Mt s enzyme cu to gm ton nhng phn t L amino acid lin kt vi nhau to thnh, gi l enzyme mt thnh phn. a s enzyme l nhng protein phc tp gi l enzyme hai thnh phn. Phn khng phi protein gi l nhm ngoi hay coenzyme. Mt coenzyme khi kt hp vi cc apoenzyme khc nhau (phn protein) th xc tc cho qu trnh chuyn ha cc cht khc nhau nhng chng ging nhau v kiu phn ng. Mt s enzyme cn ion kim loi cho hot ng nh: Cu2+ Cytochrome oxidase 2+ 3+ Cytochrome oxidase, catalase, peroxidase Fe hoc Fe + K Pyruvate kinase 2+ Mg Hexokinase, glucose 6-phosphatase, pyruvate kinase Mn2+ Arginase, ribonucleotide reductase Mo Dinitrogenase Ni2+ Urease Se Glutathione peroxidase 2+ Zn Carbonic anhydrase , alcohol dehydrogenase, cc carboxypeptidase A v B

102 Mt s coenzyme v chc nng vn chuyn nhm tng ng ca chng nh sau: Biocytin Coenzyme A 5- Deoxyadenosylcobalamin (coenzyme B12) Flavin adenine dinucleotide Lipoate Nicotinamide adenine dinucleotide Pyridoxal phosphate Tetrahydrofolate Thiamine pyrophosphate CO2 Nhm Acyl Nguyn t H v nhm alkyl in t in t v nhm acyl Ion Hydride (:H-) Nhm Amino Nhm 1 Carbon Aldehyde

6.2. C ch tc dng
Nhng quan im hin nay nhm gii thch c ch tc dng ca enzyme u cho rng khi enzyme (E) tong tc vi c cht (S) s lm gim nng lng hot ha cc phn ng ha sinh. Mun lm gim nng lng hot ha cc phn ng enzyme cn tri qua nhiu giai on trung gian v to thnh phc cht nht nh gia E v S. Khi kt hp vi phn t enzyme, do kt qu ca s cc ha, s chuyn dch ca cc electron v s bin dng ca cc mi lin kt tham gia trc tip vo phn ng s lm thay i ng nng v th nng nn phn t c cht tr nn hot ng v d dng tham gia phn ng. Vic to thnh phc hp E-S giai on u xy ra rt nhanh v rt khng bn. Do sau mt thi gian di mi c chng minh bng thc nghim. Bng chng r rng nht v s tn ti ca phc hp E-S l thnh cng ca hai nh ha sinh Nht Bn K. Iaglu v T. Ozava l tch c phc E-S trong phn ng kh amin bng cch oxy ha (loi tr nhm amine) mt amino acid dy D do oxydase xc tc. Nhn chung ta c th hnh dung c ch tc dng ca enzyme ln c cht to sn phm bng phng trnh tng qut nh sau: E+S ES P+E Giai on 1: E kt hp vi S to thnh E-S. Giai on ny xy ra rt nhanh, nh cc lin kt khng bn nh lin kt hydro, tng tc tnh

103 din, tng tc Van der Waals Mi loi lin kt i hi nhng iu kin khc nhau v chu nh hng khc nhau khi c nc. Giai on 2: Sau khi to phc, c cht c nhng bin i nht nh v mt in t, cu hnh lm c cht tr nn hot ng hn, phn ng c d dng to thnh sn phm P. Trong nhiu phn ng do enzyme xc tc c 2 hay nhiu lai c cht, v d hexokinase xc tc phn ng: ATP + glucose hexokinase ADP + glucose 6 phosphate C ch enzyme xc tc cho phn ng 2 c cht c th nh sau: a/ C ch to phc 3 thnh phn S2

b/ C ch khng to phc 3 thnh phn

y l trng hp c cht th 2(S2) ch kt hp vo enzyme ( trng thi E) sau khi P1 c to thnh.

6.3. Trung tm hot ng (TTH) ca enzyme

T kt qu nghin cu v bn cht ho hc, v cu trc trung tm hot ng , c ch tc ng, v trung tm hot ng chng ta c th c mt s nhn xt chung v trung tm hot ng nh sau: - L b phn dng lin kt vi c cht. - Ch chim t l rt b so vi th tch ton b ca enzyme. - Gm cc nhm chc ca amino acid ngoi ra c th c c cc ion kim loi v cc nhm chc ca cc coenzyme.

104

i vi E mt thnh phn: TTH ch gm nhng nhm chc ca cc amino acid nh nhm hydroxy ca serin, carboxy ca glutamic, vng imidazol Cc nhm chc ca cc amino acid c th xa nhau trong chui polypeptide nhng nh cu trc khng gian nn n gn nhau v mt khng gian. i vi E hai thnh phn: TTH cng nh trn, cc nhm chc ca cc amino acid tham gia to thnh TTH lin kt vi nhau bng cc lin kt hydro. Ngoi ra trong TTH ca loi ny cn c s tham gia ca coenzyme v c th c ion kim loi. Theo Fisher TTH c cu trc c nh, khi kt hp vi c cht to phc E-S ta c th hnh dung ging nh cha kha v kha. Ngy nay ngi ta chng minh c rng: TTH ca enzyme ch c cu to hon chnh khi c s tng tc vi c cht (thuyt tip xc cm ng ca Koshland).

6.4. Tnh c hiu ca enzyme

Ngi ta chia tnh c hiu ra lm 3 kiu: + c hiu phn ng + c hiu c cht + c hiu khng gian a/ c hiu phn ng: l biu hin ca mt enzyme ch thng xuyn xc tc cho mt kiu phn ng nht nh, v d vn chuyn hydro t cht cho (ru bc nht hay ru bc hai) n cht nhn (NAD+ hay NADP+) hay chuyn nhm amin t mt amino acid n mt ceto acid. Cc phn ng loi th nht do dehydrogenase xc tc, cn phn ng loi th hai do aminotransferase xc tc.

105 b/ c hiu c cht: Tu mc ngi ta chia thnh: c hiu tng i v c hiu tuyt i + c hiu tuyt i: Enzyme ch tc dng ln mt c cht nht nh, mt v d c tnh cht kinh in v chuyn ho tuyt i l urease, enzyme ch phn gii ure: Hng trm th nghim trn cc dn xut ca ure u cho thy chng khng b phn gii di tc ng ca urease. Thc ra ngi ta pht hin kh nng phn gii c cht hydroxyure nhng vi tc b hn khong 120 ln. + c hiu nhm tuyt i: Cc enzyme ny ch tc dng ln nhng cht c cng mt kiu cu trc phn t, mt lin kt v c nhng yu cu xc nh i vi nhm nguyn t i vi nhm nguyn t gn lin kt chu tc dng. v d : maltase ch phn gii lin kt glucosidic c to thnh t glucoside ca glucose vi -OH ca monose khc. + c hiu nhm tng i: Cc enzyme khng c nhng yu cu i vi nhm chc gn lin kt chu tc dng. v d lipase thu phn lipid. c/ c hiu khng gian: Cc enzyme ch xc tc cho mt dng ng phn no nh dng L hay dng D, dng cis hay trans m thi.

6.5. Cc yu t nh hng n tc ca phn ng enzyme

6.5.1. nh hng ca nng enzyme Trong iu kin d tha c cht, ngha l [S] >>>[E] th tc phn ng ph thuc vo [S], v= K[E] c dng y = ax. Nh ngi ta o [E] bng cch o vn tc phn ng do enzyme xc tc. C nhiu trng hp trong mi trng c cha cht km hm hay hot ho th vn tc phn ng do enzyme xc tc khng ph thuc tuyn tnh vi [E] . v

[E]
Hnh 6.1: S ph thuc ca vn tc phn ng vo [E]

6.5.2 nh hng ca nng c cht [S] Ta kho st trng hp n gin nht : ch mt c cht.

106

E v S

(1) Gi v1 l vn tc ca phn ng to thnh phc cht ES. Gi v-1 l vn tc ca phn ng phn ly phc cht ES to thnh Gi v2 l vn tc ca phn ng to thnh E v P (sn phm). v1 = k1[E][S] v-1 = k-1[ES] v2 = k2[ES] Khi h thng t trng thi cn bng ta c: k-1[ES]+k2[ES] = k1[E][S] (k-1+k2)[ES] = k+1[E][S] (2) Gi E0 l nng ban u: [E0]=[E]+[ES]=>[E]=[E0]-[ES] (3) Thay tr s [E] t (3) vo (2) ta c: (k-1+k2)[ES] = k1([E0]-[ES]) [S] k1 [E0] [S] [ES] = -------------k-1+ k2+k1[S] Nu t Km= k-1+k2/ k1 (Km: gi l hng s Michaelis Menten). Ta c : [ES] = [E0][S]/ Km+[S] Mt khc vn tc phn ng to thnh sn phm P l: V = k2[ES] Thay [ES] bng gi tr trn ta thu c: k2[E0] [S] v = ----------------- (4) Km + [S]

107 Qua y ta thy nng enzyme cng cao th vn tc phn ng enzyme cng ln. Vn tc t cc i khi ton b enzyme lin kt vi c cht, ngha l: Vmax= k2[E0] Thay vo phng trnh (4) ta c: [S] v = Vmax ---------(5) Km+ [S] Phng trnh (5) gi l phng trnh Michaelis Menten. Km gi l hng s Michaelis Menten c trng cho mi enzyme Km c trng cho i lc ca enzyme vi c cht, Km c tr s cng nh th i lc ca enzyme vi c cht cng ln, ngha l vn tc ca phn ng do enzyme xc tc cng ln.

[S]
Hnh 6.2. Bin thin vn tc phn ng theo nng c cht.

Khi tng [S] th v phn ng tng, tng [S] n mt gi tr no th v t n gi tr vmax v s khng tng na nu ta vn tip tc tng [S]. Khi Km=[S] th v =1/2 Vmax Nm 1934. Lineweaver v Burk, trn c s ca phng trnh (5) nghch o bin thnh dng ng thng y=ax+b, n c ngha ln i vi vic nghin cu km hm enzyme.

108

1/v

1/Vmax -1/Km 1/[S]

Hnh 6.3: S ph thuc ca tc phn ng vo nng c cht theo Lineweaver-Burk

6.5.3. nh hng ca cht km hm (inhibitor) L cht c tc dng lm gim hot hay lm enzyme khng cn kh nng xc tc bin c cht thnh sn phm. Km hm enzyme c th thc hin bng nhiu cch khc nhau (thun nghch hay khng thun nghch). Thun nghch c: Cch 1: Km hm cnh tranh (competitive inhibition) Trong trng hp km hm cnh tranh l c cht v cht km hm u tc dung ln trung tm hot ng ca enzyme, Cht km hm chon ch ca c cht enzyme.

Hnh 6.4. Kiu km hm cnh tranh

Khi c cht d tha, nng cht km hm thp th c th loi b tc dng ca cht km hm, cn nng c cht thp v nng cht km hm cao th li c tc dng km hm hon ton.

1/v= (Km/Vmax) 1/S +1/Vmax

= 1+[I]/KI

109 1/v

[I]

1/Vmax

Khng c cht km hm

1/[S]
Hnh 6. 5. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver-Burk khi c km hm cnh tranh

Ngi ta thy km hm nh vy phn ln xy ra gia cht km hm v c cht c s tng ng v mt ho hc. v d: malic acid c cu trc gn ging vi succinic acid nn km hm cnh tranh enzyme succinatedehydrogenase, l enzyme xc tc cho s bin i succinic acid thnh acid fumaric acid. Trng hp c bit ca km hm cnh tranh l km hm bng sn phm. Trng hp ny xy ra khi mt sn phm phn ng tc dng tr li enzyme v chon v tr hot ng phn t enzyme. ng thng c cht km hm th c xin ln hn v ct trc tung mt im l 1/Vmax Cch 2: Km hm phi cnh tranh (uncompetitive inhibition) c trng ca kiu km hm ny l cht km hm ch lin kt vi phc hp ES, m khng lin kt vi enzyme t do.

110

Hnh 6.6. Kiu km hm phi cnh tranh

1/v=(Km/Vmax)1/[S] + /Vmax
1/v [I]

-1/Km

khng c cht km hm

1/[S]
Hnh 6.7. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver-Burk khi c km hm phi cnh tranh

111

Cch 3: Km hm hn tp( mixed inhibition )

Hnh 6.8. Kiu km hm hn tp

Trong , cht km hm khng nhng lin kt vi enzmye t do m cn lin kt vi c phc hp ES to thnh phc hp EIS khng to c sn phm P. Tng t nh trn ta c phng trnh :

1/v= (Km/Vmax)1/[S] +/vmax

1/v [I]

1/Vmax

khng c cht km hm

1/[S]
Hnh 6.9. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver-Burk khi c km hm hn tp

112 Cc gi tr , c nh ngha nh trn. Trng hp = gi l km hm khng cnh tranh (noncompetitive inhibition). Mt trng hp km hm cn gp na l km hm enzyme bng nng cao ca c cht gi l km hm c cht nh km hm urease khi nng ure cao, ngoi ra cn c cc enzyme khc nh lactatdehydrogenase, carboxypeptidase, lipase, pyrophotphatase, photphofructokinase (i vi ATP). Nguyn nhn ca nhng hin tng ny cn cha c bit r. c th l: + Tn ti nhiu trung tm lin kt vi c cht bng cc i lc khc nhau. Khi nng c cht thp th enzyme c th ch lin kt vi mt phn t c cht, cn khi nng c cht cao n lin kt vi nhiu c cht dn n hnh thnh phc hp ES khng hot ng. + C cht cng c th c lin kt nh nhng v tr c bit ca enzyme. l mt nhm enzyme quan trng (enzyme d lp th) bn cnh trung tm xc tc cn c trung tm iu chnh. + C cht c th lin kt vi mt cht hot ho v bng cch ny n tch khi E. + C cht c th chon ch (ngn cn) mt cofactor ( ng yu t ) hay mt coenzyme. + C cht c th nh hng n ion lc ca mi trng v qua lm mt i tnh chuyn ho ca enzyme. 6.5.4. nh hng ca cht hot ha (activator) L cht lm tng kh nng xc tc nhm chuyn ha c cht thnh sn phm. Thng thng l nhng cation kim loi hay nhng hp cht hu c nh cc vitamin tan trong nc. V d: Mg++ hot ha cc enzyme m c cht c phosphoryl ha nh pyrophosphatase (c cht l pyrophosphate), adenosinetriphosphatase (c cht l ATP). Cc cation kim loi c th c tnh c hiu, tnh i khng v tc dng cn tu thuc vo nng . 6.5.5. nh hng cu nhit Ta c th tng vn tc ca mt phn ng ha hc bng cch tng nhit mi trng, hin tng ny tun theo quy lut Vant-Hoff. iu ny c ngha khi tng nhit ln 100C th tc phn ng tng ln khang 2 ln. i vi phn ng do enzyme xc tc cng c th p dng c quy lut ny nhng ch trong mt phm vi nht nh,v bn cht enzyme l protein.Khi ta tng nhit ln trn 40-500C xy ra qu trnh ph hu cht

113 xc tc. Sau nhit ti u tc phn ng do enzyme xc tc s gim. Nh tn ti nhit ti u ngi ta phn bit phn ng ho sinh vi cc phn ng v c thng thng. Mi enzyme c mt nhit ti u khc nhau, phn ln ph thuc ngun cung cp enzyme, thng thng trong khong t 40-600C , cng c enzyme c nhit ti u rt cao nh cc enzyme ca nhng chng a nhit. Cc chng vi sinh vt a nhit, c bit cc vi khun chu nhit c cha enzyme chu nhit cao. Hat

nhit
Hnh 6.10. nh hng ca nhit ln hat enzyme

6.5.6. nh hng ca pH S phn li khc nhau ca mt phn t protein cc gi tr pH khc nhau lm thay i tnh cht ca trung tm lin kt vi c cht v tnh cht hot ng ca phn t enzyme.iu ny dn n gi tr xc tc khc nhau ph thuc vo gi tr pH. Nh bit mi enzyme c mt pH ti u,mi enzyme c ng biu din nh hng pH ln vn tc phn ng do chng xc tc. ng biu din c dng nh hnh sau: Hat

Hnh 6.10. nh hng ca pH ln hat enzyme

114 nh hng ca gi tr pH n tc dng enzyme c th do cc c s sau: a/ Enzyme c s thay i khng thun nghch phm vi pH cc hp. b/ hai sn ca pH ti u c th xy ra s phn ly nhm prosthetic hay coenzyme. c/ Lm thay i mc ion ho hay phn ly c cht. d/ Lm thay i mc ion ho nhm chc nht nh trn phn t enzyme dn n lm thay i i lc lin kt ca enzyme vi c cht v thay i hot tnh cc i. Nh xc nh Vmax v Km ph thuc gi tr pH cho php nhn nh li bn cht ca cc nhm tham gia vo lin kt c cht v qu trnh t xc tc. 6.5.7. Cc yu t khc + nh sng: C nh hng khc nhau n tng loi enzyme, cc bc sng khc nhau c nh hng khc nhau, thng nh sng trng c tc ng mnh nht, nh sng c tc ng yu nht. nh sng vng t ngoi cng c th gy nn nhng bt li, enzyme trng thi dung dch bn hn khi c kt tinh dng tinh th, nng enzyme trong dung dch cng thp th cng km bn, tc ng ca tia t ngoi s tng ln khi nhit cao. V d di tc ng ca tia t ngoi nhit cao enzyme amylase nhanh chng mt hot tnh. + S chiu in: in chiu vi cng cng cao th tc ng ph hu cng mnh. Tc ng s mnh hn i vi dch enzyme c nng thp. C th do to thnh nhng gc t do, t tn cng vo phn ng enzyme. + Sng siu m: Tc ng rt khc nhau i vi tng loai enzyme, c enzyme b mt hot tnh, c enzyme li khng chu nh hng. Nhn xt chung: bn ph thuc vo trang thi tn ti ca enzyme, cng tinh khit th enzyme cng km bn, dch cng long th bn cng km, tc ng ca mt s ion kim loi trong dch vi nng khong 10-3M nh Ca++ lm tng tnh bn. Enzyme allosteric ( Enzyme d lp th, d khng gian) Cho n nay, ngi ta m t enzyme m hat tnh enzyme ph thuc nng c cht khng c dng hyperbol m c dng sigmoid l enzyme allosteric (Hnh 6.11 ):

115

Hnh 6.11. Bin thin vn tc phn ng theo nng c cht

i vi enzyme ny, khi nng c cht thp th tc phn ng tng chm, sau tip tc tng nng th tc nhanh chng t gi tr cc i. Nh ta bit, enzyme tun theo ng hc Michaelis-Menten th 1 hay nhiu c cht cng ch lin kt vo 1 v tr trn phn t enzyme, iu ny s dn n enzyme bo ha c cht. Cn enzyme c ng cong tc sigmoid ch xut hin khi enzyme l mt oligomer, nn c th lin kt vi nhiu phn t c cht. iu ny c ngha trn enzyme allosteric c nhiu trung tm lin kt, mi monomer c 1 trung tm lin kt. Ngi ta cho rng, trong trng hp ny c tnh hp tc gia cc v tr lin kt c cht trong phn t enzyme oligomer. Cc enzyme oligomer ny c Monod gi l allosteric enzyme d lp th (allosteric enzyme). ng cong tc sigmoid c th b cht iu ha (modulator) y v pha tri hay phi. Cht iu ha dng tc lm tng i lc ca enzyme allosteric vi c cht, ngc li l cht iu ha m. Cc cht iu ha c th lm nh hng khc nhau n cc thng s ng hc, lm thay i gi tr ring l mt trong hai gi tr Km hay Vmax.

Hnh 6.12 Minh ha khi c modulator

116

6.6. Cch gi tn v phn loi enzyme

Nh ta bit mi enzyme xc tc cho mi kiu phn ng ho hc duy nht (nh oxy ho mt kiu c cht nht nh, thu phn mt kiu lin kt nht nh,vn chuyn mt nhm cht nht nh t mt cht cho n mt cht nhn c a ch, trong c c vic bin i ch mt c cht duy nht), mt khc cn c mt kiu phn ng ho sinh nht nh c th c xc tc bng cc enzyme khc nhau. Da vo tnh c hiu phn ng ca enzyme, nm 1961 tiu ban enzyme hc quc t trnh by mt bo co, trong c ngh nhng nguyn tc nh tn v phn loi enzyme. Ngi ta chia enzyme ra lm 6 lp: 1. Oxydoreductase: cc enzyme xc tc cho cc phn ng oxi ho-kh. 2. Transferase: cc enzyme xc tc cho cc phn ng chuyn v. 3. Hydrolase: cc enzyme xc tc cho cc phn ng thy phn. 4. Lyase: cc enzyme xc tc cho cc phn ng phn ct khng cn nc. 5. Isomerase: cc enzyme xc tc cho cc phn ng ng phn ho. 6. Ligase (synthetase): cc enzyme xc tc cho cc phn ng tng hp c s dng lin kt giu nng lng ca ATP .v.v. Mi lp chia thnh nhiu t (di lp), mi t chia thnh nhiu nhm (siu lp). Tn enzyme thng c gi: Tn c cht c hiu - loi phn ng xc tc cng thm tip v ng ase. ng trc tn enzyme thng c 4 con s: s th nht ch lp, s th hai ch t, s th ba ch nhm, s th t ch s hng enzyme trong nhm. V d: (2.6.1.1) L.aspartate: -cetoglutarate aminotransferase. Enzyme ny xc tc cho phn ng chuyn nhm amine t L.aspartate n -cetoglutarate. L.aspartate +-cetoglutarate oxaloacetate + glutamate

6.7. Cc coenzyme quan trng

Nicotinamide adenine dinucleotide

117

Flavine adenine dinucleotide

Lipoic acid

Coenzyme A

Biotin

118

Thiamin diphosphate

(Nhm - amino protein, Schiff base)

Pyridoxal phosphate

Tetrahydrofolic acid

119

TI LIU THAM KHO

Ti liu ting Vit
1. Phm Th Trn Chu, Trn thi ng. 1999. Ho sinh hc, NXB Gio dc, H Ni. 2. Qu Hai. 2004. Gio trnh Ha sinh i cng, Ti liu lu hnh ni b Trng HKH Hu. 3. Trn Thanh Phong. 2004. Gio trnh Ha sinh i cng, Ti liu lu hnh ni b Trng HKH Hu. 4. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting Anh

1. Bergmeyer H. U. 1968. Methods of enzymatic analysis, translated from the third German edition, Acanamic, New York. 2. Copeland R. A. 2000. Enzymes, copyright by Wiley-VCH, Inc. 3. Lehninger A. L. 2004. Principles of Biochemistry, 4th Edition. W.H Freeman. 4. Mikkelsen S. R. 2004. Bioanalytical chemistry, copyright by John Wiley & Sons, Inc.