Chapter 8 Yonsei University Department of Biochemistry The

  • Slides: 18
Download presentation
Chapter 8. 효소반응속도론 Yonsei University Department of Biochemistry The laboratory of tumorigenesis and senescence

Chapter 8. 효소반응속도론 Yonsei University Department of Biochemistry The laboratory of tumorigenesis and senescence 서진호

Enzyme & Enzyme kinetics v Catalytic power Activation energy (Ea or ΔG‡)를 낮춤으로써 반응

Enzyme & Enzyme kinetics v Catalytic power Activation energy (Ea or ΔG‡)를 낮춤으로써 반응 속도를 증가시킴. v specificity lock & key model, induced fit model v Activity는 온도, chemical environment (p. H), substrate의 농도에 의해 영향을 받음. v Holoenzyme : apoenzyme (protein) + cofactor (metal, coenzyme) 2

Michaelis-Menten kinetics equation V 0 = Vmax [S] Km + [S] Vmax = maximum

Michaelis-Menten kinetics equation V 0 = Vmax [S] Km + [S] Vmax = maximum velocity V 0 = initial velocity [S] = substrate conc. Km = (k -1+k 2)/k 1 = Vmax /2 일 때 기질농도 * k 1 >> k-1 * steady-state assumption => [ES]는 일정 k 1[E][S] = (k -1+k 2)[ES] [E][S] / [ES] = (k -1+k 2)/k 1 4

Double-reciprocal plot Vmax [S] V 0 = 1 V 0 Km + [S] =

Double-reciprocal plot Vmax [S] V 0 = 1 V 0 Km + [S] = y = Km Vmax ax · 1 [S] + + Lineweaver-Burk plot Double-reciprocal plot 5 1 Vmax b

Competitive Inhibitor Ki = [E][I]/[EI] KMAPP 증가 : x절편 증가 Vmax. APP 변함없음 :

Competitive Inhibitor Ki = [E][I]/[EI] KMAPP 증가 : x절편 증가 Vmax. APP 변함없음 : y절편 그대로 7

Uncompetitive Inhibitor Ki = [ES][I]/[ESI] KMAPP 감소 : x절편 감소 Vmax. APP 감소 :

Uncompetitive Inhibitor Ki = [ES][I]/[ESI] KMAPP 감소 : x절편 감소 Vmax. APP 감소 : y절편 증가 8

Noncompetitive Inhibitor KMAPP 변함없음 : x절편 그대로 Vmax. APP 감소 : y 절편 증가

Noncompetitive Inhibitor KMAPP 변함없음 : x절편 그대로 Vmax. APP 감소 : y 절편 증가 9

Double-reciprocal plot for Inhibitor KMAPP= KM(1 + [I]/Ki) Vmax. APP = Vmax /(1 +

Double-reciprocal plot for Inhibitor KMAPP= KM(1 + [I]/Ki) Vmax. APP = Vmax /(1 + [I]/Ki) 10

Experimental method Enzyme : Alkaline phosphatase(ALP) optimum p. H : p. H 9 substrate

Experimental method Enzyme : Alkaline phosphatase(ALP) optimum p. H : p. H 9 substrate : ρ-Nitrophenylphosphate(PNPP) : no color product : ρ-Nitrophenol(PNP) : bright yellow , 410 nm에서 최대흡광 (ε for PNP= 18300 M-1 cm-1) *OD값=e*l*c e=몰 흡광계수, l=cuvette의 크기, c=concentration (무색) 11 (밝은 노랑)

Procedure meterials Spectrophotometer Enzyme : calf intestine alkaline phosphatase (0. 05 U / ul)

Procedure meterials Spectrophotometer Enzyme : calf intestine alkaline phosphatase (0. 05 U / ul) Substrate : ρ-nitrophenylphosphate (50 m. M) Unknown inhibitor A (50 m. M) Reaction buffer : 100 m. M Tris-HCl, 2 m. M spermidine, 0. 2 m. M Zn. Cl 2, 2 m. M Mg. Cl 2 Stop buffer : 1 M Na. OH Stopwatch D. W (distrilled water) 12

Result 1. <Determining Km value of alkaline phosphatase> 2 3 14 20 40 80

Result 1. <Determining Km value of alkaline phosphatase> 2 3 14 20 40 80 160 320 10 10 10 470 450 410 330 170

Result 2. <Determining Ki value of inhibitor> 2 3 15 20 40 80 160

Result 2. <Determining Ki value of inhibitor> 2 3 15 20 40 80 160 320 10 10 10 445 425 385 305 145 25 25 25

결과처리   1 2 3 4 5 500 500 500 20 10 40 10

결과처리   1 2 3 4 5 500 500 500 20 10 40 10 80 10 160 10 320 10 470 450 410 330 170 0 min 1 min 2 min 0. 003 0. 101 0. 129 0. 004 0. 145 0. 004 0. 09 0. 191 0. 005 0. 142 0. 269 0. 006 0. 277 0. 353 3 min 0. 134 0. 168 0. 231 0. 294 0. 4 DA/min M/min 0. 068 3. 728 E-06 0. 074 4. 029 E-06 0. 085 4. 648 E-06 0. 122 6. 655 E-06 0. 192 1. 049 E-05 [S](M/L) 0. 001 0. 002 0. 004 0. 008 0. 016 Buffer Substrate Enzyme DW 1/[S] 1/V 1000 500 250 125 62. 5 2. 682 E+05 2. 482 E+05 2. 152 E+05 1. 503 E+05 9. 534 E+04 350000 300000 추세선 그리고 추세선 서식에서 식 나타냄 체크 &250000 200000 예측으로 뒤쪽 -1000 150000 까지해서 첨부 할 것! 100000 DA/min은 0 -1, 0 -2, 0 -3까지 속도들의 평균을 구할 것 50000 0 -50000 18 0 200 400 600 800 1000 1200