C1: i cng v enzymeC2: ng hc enzymeC3: Mt s phng php nghin cu enzyme

Enzyme

Chng 1: I CNG V ENZYME1.1. Danh php v phn loi1.2. Cu to v cu trc1.3. Tnh cht v c ch tc dng1.5. Tnh c hiu ca enzyme

Cn c gi l men L cht xc tc sinh hc (biocatalysis) c bn cht l protein C trong t bo mi c th sinh vt sng Xc tc c hiu cho cc phn ng ha hc nht nh trong c th sinh vt (invivo) v ngoi t bo (invitro)nh ngha Enzyme :

C th thiu th mi qu trnh chuyn ha s b nh ch sinh vt khng th sng, sinh sn v pht trin bnh thng s sng khng tn ti Hin nay, c hn 2000 enzyme c khm ph trong c hn 200 c thu nhn dng tinh th ng dng rng ri trong nhiu lnh vc

Cc hng nghin cu chnh v enzyme:1. Nghin cu v hon thin cc phng php tch v tinh ch enzyme xc nh cu trc phn t, thu nhn ch phm c tinh khit cao 2. Nghin cu iu kin, tc tc ng cng nh cc yu t vt l v ha hc nh hng n hot ng ca enzyme. 3. Tm hiu bn cht ca qu trnh xc tc ca enzyme v c ch tc dng

Cc hng nghin cu chnh v enzyme:4. Nghin cu sinh hc enzyme 5. Nghin cu tnh c hiu ca enzyme 6. Nghin cu ci tin phng php k thut thc nghim mi ca ha l, sinh hc vo nghin cu enzyme thc y s pht trin ca enzyme hc7. Nghin cu enzyme ng dng trong thc t nhm mc ch h gi thnh, tng bn ca ch phm

Chng 1: I CNG V ENZYME1.1. Danh php v phn loi

1.2. DANH PHP V PHN LOI1.2.1. Danh php: Tn thng thng: c t lu v gi theo thi quen nh pepsin, trypsin, rennin, amylase khng ni ln bn cht xc tc Tn h thng:Vd: pyruvat- decarboxylase (kh CO2 ca acid pyruvic)

1.2. DANH PHP V PHN LOI1.2.2. Phn loi: chia thnh 6 nhm chnh Nhm 1: oxydoreductase : xc tc p oxi ha kh Nhm 2: transferase : xc tc p chuyn v Nhm 3: hydrolase: xc tc p thy phn Nhm 4: lyase: xc tc p phn ct khng cn nc Nhm 5: isomerase: xc tc p ng ha Nhm 6: ligase (synthetase): xc tc p tng hp c s dng lin kt giu nng lng ATP

Chng 1: I CNG V ENZYME1.2. Cu to v cu trc

1.2. CU TO V CU TRC1.2.1. Cu to: Enzyme mt cu t: trong thnh phn cu to ch c protein (lysozyme, pepsin) Enzyme hai cu t: Phn protein Phn khng phi protein: cc dn xut ca vitamin, kim loi nucleotit

1.2. CU TO V CU TRC1.2.1. Cu to:Vd: catalase, peroxydase, cytochrom, polyphenol oxydase .. nhm ngoi cha Cu, Fe pyruvat decarboxylase nhm ngoi l dn xut vitamin B1 amino transferase nhm ngoi l vitamin B6

1.2. CU TO V CU TRCCofactors

Mt s coenzyme thng dng

1.2. CU TO V CU TRC1.2.2. Cu trc: Cch sp xp c trng trong khng gian ca cc chui polypeptide ring bit trong phn t enzyme Trung tm hot ng(tth): L mt phn nh trong phn t enzym tham gia trc tip lin kt vi c cht, quyt nh hot tnh xc tc enzym S trung tm hot ng ca enzym c th l mt hay nhiu hn

Enzyme mt thnh phn, tth gm cc nhm chc nng ca amino acid khng tham gia to trc chnh ca si polypeptid

V d:Cysteine (Cys)Serine (Ser)Lysine (Lys)Aspartic acid (Asp)

*V d: (vng imidazol, indol)Tryptophan (Trp) Tyrosine (Tyr)12/02/2011Histidine (His)

* Nhm COOH, guanidinGlutamic acid (Glu)Arginine (Arg)12/02/2011

1.3. CU TO V CU TRCActive SerChymotrypsin

The catalytic triad

Asp102His57Ser195Catalytic TriadChymotrypsin Catalytic Mechanism A1OCheck substrate specificity

TTH ca cc enzyme 2 thnh phn thng bao gm nhm ngoi v cc nhm chc nng ca cc amino acid phn apoenzyme

Chng 1: I CNG V ENZYME1.3. Tnh cht v c ch tc dng1.3.1. Tnh cht chung ca enzyme1.3.2. Cng lc xc tc1.3.3. C ch tc dng

1.3. TNH CHT V C CH TC DNG1.3.1. Tnh cht chung ca enzyme Enzym khng thm tch qua mng bn thm. Enzym c tnh cht lng tnh. Tan trong nc, dung mi hu c c cc khc, dung dch mui long, glycerin. D b bin tnh bi nhit cao v mt hot tnh xc tc. V hnh dng a s enzym l hnh cu

1.3. TNH CHT V C CH TC DNG1.3.2. Cng lc xc tc Kh nng ca enzyme chuyn ha c cht thnh sn phm Enzyme lm gim nng lng hot ha ca phn ng xc tc

Uncatalyzed activation energy Enzymatic activation energyEnergyProgress of reactionTotal energy Changes of reactionNon-enzymatic activation energy Substrate Product ACTIVATION ENERGY

1.3. TNH CHT V C CH TC DNG1.3.2. Cng lc xc tc

1.3. TNH CHT V C CH TC DNG1.3.3. C ch tc dng

Extra Negative Charge Was NeutralizedE + SAdapted from Dressler & Potter (1991) Discovering Enzymes, p.179

Key and lockInduce fit(tip xc cm ng)

The induced conformational change in hexokinase M11.8Induced fit is the main model for changes in the enzyme to fit the transition state and to bring functional side groups of the enzyme to the right location to help the catalysis.

Basic Mechanism of Catalysis3 basic types1) Bond Strain2) Acid-base transfer3) OrientationConformational changeChemical reactionSpace arrangementCarboxypeptidase ACarboxypeptidase BCarboxypeptidase YConcertSequentialChymotrypsinTrypsinElastasenon-polarRKnon-specificYFWRKGASer-proteaseEndopeptidaseMetal proteaseExopeptidaseJuang RH (2004) BCbasics

Asp102His57Ser195Catalytic TriadChymotrypsin Catalytic Mechanism A1OCheck substrate specificity

Asp102His57Ser195HHChymotrypsin Catalytic Mechanism A2OFirst Transition State

HHChymotrypsin Catalytic Mechanism A3OAcyl-Enzyme Intermediate

HChymotrypsin Catalytic Mechanism D1OAcyl-Enzyme Water Intermediate

HChymotrypsin Catalytic Mechanism D2OHSecond Transition State

HChymotrypsin Catalytic Mechanism D3ODeacylation

Concerted Mechanism of Catalysis12345His(196)His (69)Glu(72)+Arg (145)Carboxypeptidase AC-terminusACTIVESITECheck forC-terminalSite forspecificityActivesite pocketSubstratepeptide chainJuang RH (2004) BCbasics

Zymogens Mt s enzym tiu ha nh pepsin, trypsin, chymotrypsin khi mi tit ra dng khng hot ng gi l tin enzym (zymogen). Ch hot ng khi c hot ha. Qu trnh hot ha c s ct b mt s peptid, peptid-km hm hot ng enzym hoc bao vy cc nhm chc hot ng ca E sp xp li ni ti ca phn t E hnh thnh tm hot ng ca E v c kh nng hot ng.

Zymogens

Chymotrypsin Is Activated by ProteolysisAdapted from Campbell (1999) Biochemistry (3d) p.179245R15-I16Chymotrypsinogen (inactive)p-Chymotrypsin (active)S14-R15T147-N148Trypsina-Chymotrypsinogen (active)p-ChymotrypsinI16L13A149Y146Disulfide bonds

Chng 1: I CNG V ENZYME1.5. Tnh c hiu ca enzyme1.5.1. c hiu kiu phn ng1.5.2. c hiu c cht

1.5. TNH C HIU CA ENZYME

V d 1: enzyme sucrase xc tc cho phn ng phn hy sucrose thnh glucose v fructose

Prentice-Hall 2002General Chemistry: Chapter 28Slide * of 57SUCRASE MECHANISM

Prentice-Hall 2002General Chemistry: Chapter 28Slide * of 57SUCRASE MECHANISM

V d 2: urease xc tc phn ng phn hy ure

V d 3: fumarate hydratase xc tc phn ng thy phn L-malic acid thnh fumaric acid:

Specificity of Ser-Protease FamilyActive SiteTrypsinChymotrypsinElastasecut at Lys, Argcut at Trp, Phe, Tyrcut at Ala, GlyNon-polarpocketDeep and negativelycharged pocketShallow andnon-polarpocketJuang RH (2004) BCbasics

*P c xc tc*E5-18Chymotrypsin carbonyl (C=O) -O: () Chymotrypsin (1) Ser -O: (2) Nng lng hot ha l mc nng lng cn thit chuyn cc phn t cht tham gia phn ng t trng thi bnh thng sang trng thi hot ng- phn ng xy**Qu trnh to phc cht enzym-c cht v bin i phc ny thnh sn phm, gii phng enzym t do thng tri qua 3 giai on:GD1: enzyme kt hp vi c cht bng lin kt yu to thnh phc hp enzyme - c cht (ES) khng bn, phn ng ny xy ra rt nhanh v i hi nng lng hot ha thp; GD2: xy ra s bin i c cht dn ti s ko cng v ph v cc lin kt ng ha tr tham gia phn ng. GD3: to thnh sn phm, cn enzyme c gii phng ra di dng t do.

*E5-22Chymotrypsin -N-H () Chymotrypsin *E5-3Hiu ng orientation l g ?E5-18Chymotrypsin carbonyl (C=O) -O: () Chymotrypsin (1) Ser -O: (2) E5-18 E5-18 E5-18 E5-18 E5-18 E5-4 carboxypeptidase (1) Zn2+ carbonyl (2) (3) Glu 270 OH- C+ (2) C-OH (4) Tyr 248-OH lone pair (5) C- R Arg 145 C- -COOH C-Zymogen: proenzyme*Gastric: thuc v d dyPancreas: tuyn ty**Proteolysis: s phn gii protein u tin enzyme chymotrypsinogen c sn sinh tuyn ty. Sau tin enzyme ny c hot ha bng cch s dng trypsin loi b lin kt peptide gia Arg-15 v Ile-16 to thnh -chymotrypsin.-chymotrypsin loi b 2 dipeptide (14-15 v 147-148) ca nhng -chymotrypsin khc to thnh -chymotrypsin. Hot tnh ca -chymotrypsin vn c gi nguyn nh hot tnh ca -chymotrypsinE5-8Do cu trc l ha c bit ca phn t enzyme v c bit l ca trung tm hot ng m enzyme c tnh c hiu rt cao so vi nhng cht xc tc thng thng khc. Mi enzyme ch c kh nng xc tc cho s chuyn ha mt hay mt s cht nht nh theo mt kiu phn ng nht nh. c tnh tc dng la chn cao ny gi l tnh c hiu hoc tnh chuyn ha ca enzyme. Tinh c hiu l mt trong nhng c tnh c bn quan trng nht ca enzyme*Mi enzyme ch xc tc cho s chuyn ha mt hoc mt s cht nht nh. Mc c hiu c cht ca cc enzyme khc nhau khng ging nhauc hiu tuyt i: Mt s enzyme hu nh ch xc tc cho phn ng chuyn ha mt c cht hoc mt nhm c cht xc nh v ch xc tc cho phn ng y m thi. Chng cng c th phn gii mt vi cht khc nhng vi vn tc thp hn nhiu. Nhng enzyme c tinh c hiu tuyt i thng c dng nh lng chnh xc c cht ca n.Vd: urease thy phn ure, maltase: thy phn lin kt glucosesidec hiu tng i: Enzyme co kh nng tac dng len mt kiu lien kt hoa hc nht nh trong phan t c cht ma khong ph thuc vao cu to ca cac phn tham gia to thanh mi lien kt oc hiu quang hc: Hu nh tt c cac enzyme u co tinh c hiu khong gian rt cht ch, ngha la enzyme ch tac dng vi mt trong hai dng ng phan khong gian ca c cht*Invertases and sucrases hydrolyze sucrose to give the same mixture of glucose and fructose. Invertases cleave the O-C(fructose) bond, whereas the sucrases cleave the O-C(glucose) bond.*Sucrase l enzyme xc tc phn ng phn gii sucrose thnh gluco v fructose*E5-26Ser Trypsin Asp 189 Lys Arg