Lecture 1. Introduction to Biochemistry

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1 Chapter 7. 효소의작용기작과조절 The Behavior of Proteins: Enzymes Mechanisms and Control

2 효소반응속도의조절 Reaction Conditions No catalyst Platinum surface Catalas e Activation e nergy (kj/mol) (kcal/mol) Relative rate* x x 10 8 * Rate s are give n in arbitrary units relative to a value of 1 for the uncatalyzed reaction at 37 캜 효소는반응속도를최대 만큼가속시킨다. 효소반응은가장효율적인화학적촉매에의한반응보다 10 배에서 10,000 배까지그효율이높다 ( 없는것보다 ~10 16 ). 효소는단백질이기에온화한 (mild)) 조건에서반응해야하며효소단백질의정교한구조와기질구조의정교한물리화학적작용에의해서특이성및효율을얻는다. 효소반응의조절 - 비가역적변화 : 단백질제한효소 (protease) 에의한절단 (zymogen active enzyme) - 가역적변화 : 효소의가역적변화 - allosteric 조절 : multi-subunit enzyme (MM 기전을따르지않음 ) - 인산화 / 탈인산화 (phosphorylation/dephosphorylation) - 아데닐화 (adenylation)/ - 파레닐화 (farensylation) - 산화 - 환원반응 (oxidation-reduction); cystein-cystine

3 Types of Covalent Modification

4 Allosteric Regulation ( 다른자리입체성조절 ) - Allosteric: Greek allo + steric, other shape -allosteric enzyme : 다량체효소로효소활성이다른물질의결합에의해조절 ( 특정대사중간체등의농도변화에대하여순간적으로조절 ) - allosteric effectors : inhibitor & activator - 원핵및진핵세포. - cooperiativity ( 협동성, Hill 상수 (n) 로구분가능 ) n=1 non cooperativity n>1 positive cooperativity n<1 negative cooperativity - 예 ) PFK, Aspartate transcarbamoylase (ATCase)

Allosteric Regulation: PFK(phosphofructokinease) 의예 -glycolysis 의 rate limiting step 조절 -4 량체 : -Allosteric enzyme (4 identical) - AMP 존재시 : hyperbolic F-6-P 결합 - ATP 존재시 : sigmoidal F-6-P 결합 - Hill

5 Allosteric Regulation: PFK(phosphofructokinease) 의예 -glycolysis 의 rate limiting step 조절 -4 량체 : -Allosteric enzyme (4 identical) - AMP 존재시 : hyperbolic F-6-P 결합 - ATP 존재시 : sigmoidal F-6-P 결합 - Hill 상수 : n ~ 4 - Monod wyman : symmetry model (1965) - Substrate : F-6-P - Allosteric effectors - inhibitors : PEP, ATP - activators : ADP, AMP.GDP - two conformations : T form (tight) R form (relexed) -MRC ( 영국 ) philip evans : X-ray 구조단핵세포 : B S PFK (+substrate) E coli PFK(- effectors)

6 ATCase (aspartate carbamoyltransferase) : allosteric regulation 의다른예 - transfer of carbamoyl group (NH2 -CO- ) to asparatate - pyrimidine 합성의첫번째과정 ((UTP, CTP, TTP) - 6C6r = 12 량체 - inhibitors : CTP, UTP.. (feedback inhibition, 되먹임저해 ) - activators : ATP

7 ATCase (aspartate carbamoyltransferase) -6C6r = 12 량체 - inhibitors : CTP, UTP.. (feedback inhibition) - activators : ATP - 6C6r + Hg (p-hydroxymercuribenzoate) ---> (3C )* 2 + (2r)* 3 * allosteric site 는 active site 가아님이증명.

8 ATCase undergoes drastic structural changes upon modulator binding T = tense R = relaxed CTP = inhibitory modulator ATP & asp = stimulatory modulators

9 Allosteric activator shifts the equilibrium to the R state

10 Allosteric inhibitor shifts the equilibrium to the T state

11 Hill plot & coefficient - Hill 상수 : cooperativity의척도 예 ) PFK; n = 4 (subunit 4개 ) Hb; n = 2.8 (subunit 4개 ) n은 hill 상수 n=1 non cooperativity n>1 positive cooperativity n<1 negative cooperativity -Hill plot: a plot displaying information on site-site interaction 표현 : log y /1-y vs log[ s] log bound/unbound Log (Y /1-Y) = log([ S] n /K d )= nlog[s] logk d (Hill plot)

12 Hill plot 의유도 E + ns <-----> E Sn 해리상수는, Kd = [ E] [ S] n / [ ESn] : [ ESn] = [ E] [ S] n / Kd 기질에결합된효소의분율을 "Y" 라하면, 결합된분율, Y (bound fraction) Y = [ ESn] / [ E] +[ ESn] = ([ E] [ S] n / Kd) / ([ E] +[ E] [ S] n /Kd ) = ([ E] [ S] n ) / ([ E]*Kd +[ E] [ S] n ) = [ S] n / (Kd + [ S] n ) 결합된분율 (Y)/ 결합이안된분율 (1-Y) 로표시해보면, Y/1-Y = ([ S] n /Kd + [ S] n ) / (Kd / Kd+[ S] n ) 양변에로그를취하면, Log (Y /1-Y) = log[ S] n /Kd = nlog[ S] - logkd Hill plot ( 기울기가 Hill 상수 ) Log[S]

13 효소는아니지만 allosteric 조절의예 :Hemoglobin Myoglobin :monomer. No allosteric effect. Hemoglobin :tetramer(a 2 b 2 ) allosteric protein

14 Myoglobin 의산소결합곡선 Myoglobin 의산소결합 : MbO fractional saturation Y Y Y re arrange log( ) log 1 Y Y Mb O, equilibrium dissociation constan t K po 2 MbO2 MbO Mb 2 log K log(y/1-y) 0.0 O O 2 2 K Hill Plot Mb O 2 MbO 2 slope=1 P 50 (K) po 2 (torr) log P 50 log po 2

15 Hemoglobin 의산소결합곡선 : (cooperative binding, sigmoidal curve) Hb 에 4 분자의산소결합 : Hb( O ) fractional saturation Y Y Y re arrange log( ) 4log 1 Y Y Hb 4O 2, equilibrium dissociation po 2 Hb( O2 ) 4 Hb( O ) Hb 2 log K 4 log(y/1-y) 0.0 constan t K O O K Hill Plot slope=4 4 Hb O2 Hb( O ) 2 4 log P 50 P 50 po 2 (torr) log po 2

16 Hemoglobin 의산소결합곡선 : (cooperative binding, sigmoidal curve) Hb 는실험에의하면 un-coorperative (Myoglobin 처럼 ) 과 completely cooperative binding 의중간형태혹은혼합형태를보인다. 실제로측정하면 Hb 의산소결합의 Hill coefficient 는약 2.8 (<4) 로이론적최대치인 4 보다는작다. Y 0.5 n=4 n=2 log(y/1-y) 0.0 Hill Plot slope=1 slope=2.8 slope=1 P 50 po 2 (torr) log K 4 log K 1 log po 2

17 P50 :the point of half-saturation. po2 : 산소분압

18 Hemoglobin 활성조절의목적 산소에대한생리적필요를충족 : Release O 2 Bind O 2

19 다른자리입체성효소작용모델 : 협주적 & 순차적 The Concerted Model ( 협주적모델 ) Wyman, Monod, and Changeux 1965 효소는 2 가지구조로존재 : R (relaxed): 강한기질결함, 활성형태 ( 쌍곡선 ) T (tight or taut): 약한기질결합, 비활성상태 (sigmoidal) 기질의부재시대부분의효소는 T (inactive) 상태로존재 기질의결합 : T (inactive) R (active) 전환 T >R 전환 : subunits 구조는동시에변화 allosteric effector: 단백질 ( 효소 ) 의 4 차구조에영향을주는물질 homotropic effects: 동일한물질들의결합에의한효과를주는것예 ) aspartate 의 ATCase 결합 heterotropic effects:; 다른물질들의결합에의한효과를주는것예 ) ATCase의 CTP에이한저해그리고 ATP에의한활성

20 The Concerted Model ( 일치형 ( 협주적 ) 모델 ) two subunits 단백질의예 Subunit 모두동시전환 : T to R 전환시 (Figure 6.4(a) 1965: Monod-Wyman-Changeaux ( 쌍줴 ) model)

21 The Concerted model for allosteric proteins (described by Monod, Wyman and Changeux) (aka MWC model) T-state R-state ATCase follows this model better

22 Sequential Model ( 순차적모델 ) 1966: Koshland 기질의결합 : T R form 으로순차적구조전환유도 구조의변화는기질의결합에의해유도됨 (induced fit).

23 Kinetics of allosteric enzyme (2 개의기질인경우 ) ( 유도식 ) K1 = [ E] [ S] / [ ES] = [ E] [ S] /[ SE] (:[ SE] =[ ES] ) [ES] = [ SE] = [ E] [ S] / K1 K2 = [ ES] [ S] / [ SES] [SES] = [ ES] [ S] / K2 = [ E] [ S] 2 / K1 K2 ( 여기서 K1, K2 는해리상수 ) K1 & K2 : 해리상수 a) 반응의속도 V, ( 기질두개 : 간략히 k2 ~ Kcat 라면, 여기서 k2 는속도상수 ) V = Kcat ([ ES] +[ SE] + 2[ SES] ) = Kcat ([ E] [ S] /K1 + [ E] [ S] /K1 + 2 [ E] [ S] 2 /K1 K2 ) = 2Kcat ([ E] [ S] /K1 + [ E] [ S] 2 / K1 K2 )

24 Kinetics of allosteric enzyme (2 개의기질인경우 ) ( 유도식계속 ) b) 반응의최대속도, Vmax 는 Vmax = 2Kcat [Et] = 2Kcat [ E] + [ E] [ S] /K1 + [ E] [ S] /K1 + [ E] [ S] 2 /K1 K2] 여기서, 효소의총농도 [Et] 는, [Et] = [ E] +[ES] +[SE] +[SES] c) V / Vmax 는? V / Vmax = 2Kcat ([E] [ S] /K1 + [ E] [ S] 2 /K1 K2) Kcat ([ E] +[ E] [ S] /K1 +[ E] [ S] /K1+[ E] [ S] 2 / K1 K2) 다시정리하면, V / Vmax = ([ S] /K1 + [ S] 2 /K1 K2) (1+[ S] /K1+[ S] /K1+[ S] 2 / K1 K2) = ([ S] /K1)* (1+[ S] /K2) (1+[ S] /K1) + ([ S] /K1)*(1+[ S] /K2))

25 Kinetics of allosteric enzyme (2 개의기질인경우 ) ( 유도식계속 ) V / Vmax = ([ S] /K1)* (1+[ S] /K2) (1+[ S] /K1) + ([ S] /K1)*(1+[ S] /K2)) a) K1= K2 = Kd 이면, V/Vmax = [ S] /K [ S] /K1 = [ S] K1+[ S] = [ S] Kd+[ S] 기질이두개여도, hyperbolic 결합. b) K2 <<[ S] <<K1 V / Vmax = [ S] 2 /K1K ([S]/K1 ~ 0) ( 1+ ([ S] /K1)*([ S] /K2)) (1 + ([S]/K1) * (1+[S]/K2)) = [ S] 2 /K1K [ S] 2 / K1K2 = [ S] K1K2+[ S] 2 두기질의결합력의차가있을때는, sigmoidal binding

26 Allosteric enzyme: SUMMARY Allosteric enzyme is the enzyme chosen to act as an on/off switch in the beginning of a biochemical pathway Homotrophic: modulator = substrate Heterotrophic: modulator NOT substrate Binding of modulator causes (usually) changes in quaternary structure in a cooperative fashion Allosteric enzyme switches between Tense and Relaxed states Thus, allosteric enzyme doesn t follow Michaelis-Menten kinetics

27 효소반응조절 : 비가역적활성조절 (serine protease 의예 ) serine protease : - 펩타이드결합이나 ester 결합을가수분해하는효소. - serine 이 nucleophilic 반응의중심아미노산. - 특히식이성단백질의분해에큰역할. - 예 ) trypsin, chymotrypsin, elestase - 비활성 zymogen 으로생성. 비가역적 proteolytic cleavage 로활성획득. - zymogen 의활성자리 (active site) 는구조적 distortion 에의해활성결여. 활성획득 (gain of activity) : irreversible, covalent modification by partial proteolysis zymogen (proenzyme, 전구효소, 비활성 ) > enzyme ( 활성 ) 절단 * zymogen(=proenzyme, 효소전구체 ): 촉매활성을지니지않는활성화전단계상태의효소. 구조변이에의해활성을갖게된다. 예 ) chymotrypsinogen(inactive) + 절단 (trypsin) --> chymotrypsin (active) trypsinogen (inactive ) + 절단 (enterokinease) --> trypsin (active)

-Blood-coagulation cascade 의예, Blood clotting enzymes : 7 종의 serine protease

28 Irreversible modification 의예 -chymotrypsin의활성획득 trypsin : Arg15--Ile16 절단 chmotrypsinogen (zymogen) > chymotrypsin(enzyme) -Blood-coagulation cascade 의예, Blood clotting enzymes : 7 종의 serine protease Factor X > Xa Prothrombin > thrombin Fibrinogen > Fibrin(clotting)

29 Zymogens: Chymotrypsinogen 의예 Zymogen: 효소의비활성형태 ( 전구물질 ) Chymotrypsinogen 췌장 (pancreas) 에서합성 245 amino acid residues 5 개 disulfide (-S-S-) bonds 소장으로분비시 trypsin 이 zymogen 을절단하여 chymotrypsin 으로만든후다시자신에의해절단되어 (self digestion) 으로성숙된 a chymotrypsin 형성. 1 차구조의변화후새로운 3 차구조형성 ( 활성획득 : 비가역적 )

30 Zymogens:Activation of chymotrypsin

31 serine protease 의구조 ( 세린단백질분해효소 ) -serine protease간아미노산배열과 3차구조의유사성동일한아미노산 : 빨간색 ; 절단부위 : 괄호 ; disulfide Bond : 연결선효소활성에중요한아미노산 (His, Asp, Ser) : 붉은원 HIV protease : 아스팔틱단백질분해효소파파인 : 시스테인단백질분헤효소

32 효소의기전과 Active Site 효소기전연구의방법 : 간단한 model 화합물이용 Step 1 E-OH + Enzyme O O 2 N OCCH 3 p-nitrophenylacetate O E-OCCH 3 An acyl-e nzyme inte rmediate + O 2 N O - p-nitrophenoxide O Step 2 E-OCCH 3 + H 2 O E-OH + O CH 3 CO - 효소반응작용의관심사항 : 1. 효소반응의주요아미노산은? 2. 중요아미노산의공간적배향은? 3. 효소반응의기전은? (Chymotrypsin 의효소기전의예, Ser, Phe & Tyr 이중요

Chymotrypsin 의효소기전의예 : active site residue 발견 DIPF은 chymotrypsin의 serine-195와결합하여불활성화 : 즉 Ser-195는 active site에있다는실험적증명 (peptide mapping) O Enz -CH 2 OH +

33 Chymotrypsin 의효소기전의예 : active site residue 발견 DIPF은 chymotrypsin의 serine-195와결합하여불활성화 : 즉 Ser-195는 active site에있다는실험적증명 (peptide mapping) O Enz -CH 2 OH + F-P- OCH( CH 3 ) 2 Se rine-195 OCH( CH 3 ) 2 Diisopropylphos phofluoridate (DIPF) O Enz -CH 2 O-P- OCH( CH 3 ) 2 OCH( CH 3 ) 2 A labeled enzyme (inactive)

34 효소의기전과 Active Site: Chymotrypsin 의효소기전의예 TPCK 은 chymotrypsin 의 His-57 과결합하여불활성화 : 즉 His-57 는 active site 에있다는실험적증명 C 6 H 5 CH 2 -CH- C-CH 2 Cl NH O T syl N-Tosylamido-L-phe nyle thyl chlorome thyl ketone (TPCK) (Tsyl = tos yl group) + Enz -CH 2 N N H Histidine-57 Enz -CH 2 N N C 6 H 5 CH 2 -CH- C-CH 2 NH O T syl 또한 ser-195 와 His-57 모두효소활성에중요하므로, 이두아미노산은서로근처에있음을나타낸다.

35 Chymotrypsin : Mechanism of action chymotrypsin F, Y or W 후 His- 염기 Ser- 친핵성공격 His- 산 물 - 친핵성공격

36 Catalytic Mechanisms Lewis acid/base 반응 Lewis acid: an electron pair acceptor (Mn 2+, Mg 2+, and Zn 2+ ) Lewis base: an electron pair donor ( 앞의 His residue)) 친핵성물질 : 양전하혹은전자부족극성자리공격 친전자성 : 음전하혹은전자가많은극성자리공격 많은효소의경우루이스산 / 염기조절관측 ( 예 : carboxypeptidase A Zn 2+ 포함 )

37 Catalytic Mechanisms General acid-base catalysis: proton transfer reactions Type of Group sulfhydryl amino carboxyl hydroxyl imidazole phenol Example of a Proton Donor Cys-SH Cys-S - + Lys- NH 3 Lys- NH 2 Glu-COOH Glu-COO - Ser-CH 2 OH Ser-CH 2 O - H His- CH 2 + N His- CH 2 NH Example of a Proton Acceptor T yr OH T yr O - N NH

38 효소반응의특이성 (Enzyme Specificity) 절대적특이성 (Absolute specificity): 한종류의특이한구조만결합 (lock and key model) 상대적특이성 (Relative specificity): 구조적유사한물질과결합 (induced fit model) 입체특이성 (stereospecificity): 한종류의광학적활성형태에만결합. 예 : cis alkene 의수화 (trans isomer 는결합못함 ) ( 입체적특이성 )

Catalytic Mechanisms 효소의촉매작용 (Enzyme catalysis) 반응의활성화에너지를낮추는다른경로로진행 transition 상태는기질과생성물과는다른구조 Transition state analog: 전이상태유사물 ( 반응기전연구에사용 ) Willam Jenks (1969) 가설 :

39 Catalytic Mechanisms 효소의촉매작용 (Enzyme catalysis) 반응의활성화에너지를낮추는다른경로로진행 transition 상태는기질과생성물과는다른구조 Transition state analog: 전이상태유사물 ( 반응기전연구에사용 ) Willam Jenks (1969) 가설 : 만약항체생산을유도하는면역체가반응의전이상태구조와유사하면촉매기능을갖는항체생산이가능하다주장. 화학반응시의 transition state 를인식하는항체를이용하여항체가반응을촉매하는효소기능을갖게함. Lerner & Schultz (1986) : 첫번째효소항체 (abzyme) 제작 (monoclonal antibody with catalytic activity) If an antibody is developed to a stable molecule that's similar to an unstable intermediate of another (potentially unrelated) reaction, the developed antibody will enzymatically bind to and stabilize the intermediate state, thus catalyzing the reaction. Transition state analog 프롤린라시마제반응 Pyrrole-2-carboxylate

40 Catalytic Antibodies (Abzymes) Abzyme:Transamination 반응의촉매 O P-OCH 2 COO - NHCHCH 2 CH 2 CH 2 CH 2 NH 2 CH 2 OH P H COO - + C NH 3 CH 3 D-Alanine + CH P-OCH 2 OH + N CH 3 H Pyridoxal 5'-phosphate + N CH 3 H transition state analog for the role of pyridoxal phosphate in trans amination ( 위의안정된기질유도체에대해 antigen( 항체 ) 생성 =abzyme COO - C CH 3 O Pyruvate + Abzyme NH 3 + CH 2 P-OCH 2 OH + N CH 3 H Pyridoxamine 5'-phosphate

41 보조효소 (Coenzymes) Coenzyme: 효소의일부로효소반응에참여하는 nonprotein molecule 혹은 ion ( 재생됨 ). metal ion/ organic compounds ( 주로 vitamins) Metal Ion Fe 2+ or Fe 3+ Cu 2+ Zn 2+ Mg 2+ Mn 2+ K + Ni 2+ Mo Se Enzyme Peroxidase Cytochrome oxidase DNA polyme rase He xokinase Arginase Pyruvate kinase Ure ase Nitrate reductase Glutathione peroxidase Table 6.1 Coenzymes, their reactions, and precursors Coenzyme Reaction Type Vitamin Precursor Biotin Coenzyme A Flavin coenzyme s Lipoic acid Nicotinamide adenine coenzymes Pyridoxal phosphate Tetrahydrofolic acid Thiamine pyrophosphate Carboxylation Acyl transfer Oxidation-reduction Acyl transfer Oxidation-reduction Transamination One-carbon transfe r Aldehyde transfer Pantothenic acid Riboflavin (B 2 ) Niacin Pyridoxine (B 6 ) Folic acid Thiamine (B 1 ) *Vitamin C : proline hydroxylase

42 효소반응의측정 : 빠른반응 - 빠른효소반응의측정 (steady-state 도달전 ): stopped-flow system : rapid kinetic measurement (~ 1 micro s) 반응시작 : 주사기주입 ; 관측 : 형광이나 UV-VIS

43 NAD + /NADH : 산화 - 환원반응의조효소 Nicotinamide adenine dinucleotide (NAD + ) The plus sign on NAD+ represents the positive charge on this nitrogen O O CNH 2 Nicotinamide, de rived from niacin - O- P-O- CH 2 O AMP H H HO O H OH N H + a b-n-glycosidic bond O CNH 2 H H O CNH 2 N R + + H e - NAD + NADH (oxidized fo rm) (reduced form) 340 nm 흡수없음 340 nm 흡슈 N R

44 효소반응의측정 : 일반 ( 느린 ) 반응 PFK 의 coupled assay 의예 일반적효소반응 : 반응물의감소, 혹은생성물의생성속도를 UV-VIS, 방사능, 형광등의변화관측 ( 연결된다른반응의생성물관측 )

45 연습문제 1 1) x, y 축은무엇을나타내나? 2) YO2 = 0.5의의미는? 3) Mb와 Hb 곡선모양이서로다른이유의생화학적설명은? 단, 0-25, 25~100, 100 torr 이상, 세구간을구분하여설명하시오.

46 연습문제 2 Log (Y /1-Y) = log[ S] n /Kd = nlog[ S] - logkd Hill plot ( 기울기가 Hill 상수 ) 1) Hb 산소결합의 log(y/(1-y)) 를유도하시오 2) 다음자료 (A & B 단백질 ) 를보고답하시오. a) O2 결합곡선을그려보시오. b) Hill plot 을그려보시오. c) Hill 상수를구하시오. d) 결합기전의차를설명하시오 (cooperativity) Log[S]

47 연습문제 3 ATCase 와 Hill 상수에관한물음이다. a)hg (p-hydroxymercuribenzoate) 를사용하여 ATCase 의 allosteric site 는 active site 가아님을증명할수있는방법을생각해간략히기술하시오. b) 다음그림은 effector(atp, CTP) 들의존재시 Hill equation 을사용하여물음에답하시오 : Log (Y /1-Y) = nlog[ S] logk d ( 여기서 Y 축을 log(y/1-y) 로볼수있음.) b-1) ATP,CTP 의존재및부재시 ACTase 의 Hill 상수를구하고그의미를쓰시오. ( 계산과정 ) b-2) ATP,CTP 의존재및부재시 ACTase 의기질에대한 K d 를구하시오. b-3) 어느것이 allosteric inhibitor 인가? 그이유는?

48 ATCase (aspartate carbamoyltransferase) -6C6r = 12 량체 - inhibitors : CTP, UTP.. (feedback inhibition) - activators : ATP - 6C6r + Hg (p-hydroxymercuribenzoate) ---> (3C )* 2 + (2r)* 3 * allosteric site 는 active site 가아님이증명.

49 a) 답 ) 다량체를 몇개의중합체로분해후기능분석 b) b-1) 기울기가 Hill 상수이므로 n=1 (effector 없을때와 ATP 존재시 ) n=2 (CTP 존재시 ) 의미 : CTP 존재시기질결합에대한 cooperativity 증가 b-2) 답 ) Y 축과의만나는절편이 logk d 이므로 (-) 와 ATP 존재시의 Y 절편은약 0.7 CTP 존재시는외연장 (extrapolation) 하여 Y- 절편을 구한후계산.

50 연습문제 4 Chymotrypsin 에관련된다음물음에답하시오. a) chymotrypsin 에 diisopropylphosphofluoridate(dipf) 를처리하면 27 개의 serine 가운데단지 serine 195 에만 covalent modification 이일어난다. 그이유를설명하시오. 나 ) 또한 chymotrypsin 에 tosyl-l-phenylalanine chlorometyl ketone(tpck) 를처리하면단지 histidine 57 만이 alkylation 된다. 그이유를설명하시오. 다 ) 가 ) 와나 ) 의경우 chemostrypsin 은효소활성을잃게된다. 이를근거로 chemotrypsin 의 catalytic metabolism 을유추하여보시오.

51 연습문제 5 6 장보충문제 아래의그림은효소반응의실제 data 로생성물의생성량 (Y 축 ) 과시간 (X 축 ) 의그래프이다. 사용된기질의농도는각각 10mM, 50mM 그리고 100mM 이었다. a) 어느곡선 ( 상, 중, 하 ) 이각기질농도 (10, 50, 100mM) 에해당하나? 그이유는? b) 각기질농도에대한초기속도 (Vo) 들을구하시오초기속도는직선구간만고려 ( 여기서는대략 60 초 ) c) 시간이경과함에따라속도가감소하는이유는 d) 위의자료를이용하여 [S] vs [Vo] 곡선을그리시오. e) 1/[Vo] vs 1/[S] 곡선을그리고 Km 과 Vmax 를구하시오. f) e) 에서그린그래프용지에경쟁적저해재가존재할때의가상적그래프를점선으로그려보시오. g) 만약기질의농도가 [S]=Km 이라면어떤곡선형태이겠나? 위그림에점선으로그려보시오. h) 만약반응용액의부피가 1L 이고효소의분자량이 10,000g/mol 이라면이반응의전환수 (turnover number) 는? 단효소는 1mg 존재

52 문제 6

54 문제 7

PowerPoint Presentation

PowerPoint Presentation 11 강 CHAPTER 6 Enzymes Allosteric enzyme 과반응속도론 보조효소와 vitamin 효소반응과대사의조절 불활성상태 기질 Allosteric enzyme ( 다른자리입체성효소 ) 는다중단백질소단위체로기본단위체에 활성 Subunit 조절 Subunit 조절자 Effector 기질이결합하면인접한기본단위체의촉매부위가활성화되는효소를말하며생화학