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  • Handout 4 - Schuler Ch5 5343

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    Dinesh Sd
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    kinetics

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    Handout 4- Schuler Ch5 5343

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    16 Ansichten9 Seiten

    Handout 4 - Schuler Ch5 5343

    Hochgeladen von

    Dinesh Sd
    kinetics

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    © All Rights Reserved
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    ne Nicotinamide adenine dinucleotide ioe se syne ‘Nicocinamide adenine dinucleotide tere a com wee | Eee : Ranomacleoide rated: o Scere, em Ps ferses. Tramin pyrophosphate dl 7a (Cocnayme A. siete Phospbosrancterases Cher Fale .- em rm Cytochromes Pete m=, Sesto Pyridoual phorphane ioe Pes Tetrahydrofotate coenzymes. “Somer = SoS oar oa remo ate Pye a 7 we eis Me a mente APs ho vies Ra ge) ‘Wi permison mA, Ling Boles, 25, Wooh Paha Now Yo os 3.3, ENZYME KINETICS 3.3.1. Introduction eae ep Of the ey Emymes Chap. 3 0 Figure 32, sant cone Oish=Km Substrate, (8) Siete efncnoneara Wis assumed thatthe ES complex is established rather rapidly and the rat ofthe re ‘verse reaction ofthe second step is negligible, The asumpsion of an ireversibe second ‘ection often holds only when produet accumulations neplgile a the beginning ofthe feaction. Two major approaches used in developing a rte expression forthe enzyme: ‘atlyzed reactions ar (1 apid- yes high affinity for he subst. Aso, K; eoesponds othe subst concen ton, ging the hlfemasima ection veloc ‘An equation of exactly the some form ase. 3.8 can be derived witha diferent smore general assumption applied tothe reaction scheme ine, 3. 33.22, The quasi-steady-state assumption. In many cases the assumption ‘of rapid equilibrium following mass-ation kines is not vali, although the enzyme substrate reaction sil shows saturation-4ype kine GE. Beggs and J.B. S. Haldane rst proposed using the quasi-stady-sete a sumption. In most experimental systems a closed system (atch reactor) is used in which the inital substrate concentration greatly exceeds te inital enzyme concentration, They suggest that since [Eq] was small, [ESV = 0. (Tis loge is awed. Do you see why?) CCompuer simulations ofthe actual time couse represented by eqs. 3.2, 3.3, and 3.4 have shown that ina closed system the quas-steadstate hypothesis holds afer a brief wan- sient So] > [El (for example, 100%). Figure 3.4 displays one such ime couse, By applying the quast-scady-stae assumption 09.33, we find e5}= AHELSI kathy cS) Subang the enzyme conervaion eq. 34 in cq. 39 yes = AulEa}=1ESDIS) tes) ~Aultal=IEs 619 e Eneymes chop. 3 Concentration Fgere34, Tine com f he fron ‘orca pe ni ‘sompie wlan of dt bined ‘Slevin on tis nee The fra th died box top i \ ‘ne nso frmon te over fra (thomson aed a. (igs Bachem, 2 Wer ab Time Fas ow sk 75.190) Solving ea. 3.10 for [ES]. Substituting eq 3.11 ito 09.3.2 yields yo APL BIEgISI de Gur») G12) where Ky is (+ AK and Vg is (Exh. Under most circumstances (simple exper tment) ivi impossible to detemine whether K, oF Kis more suitable. Since K, results ftom the more general derivation, we will use iti the fest of ou discussions See.39 Enzyme Kinetics 3.33. Experimentally Determining Rate Parameters ‘The determination of values for Ky ad Vj with high precision canbe difficult. Typically, experimental data are obtained from inutial-rate experiments. batch reactor is charged with a known amount of subsite (Sq) and enzyme [E3. The product (or substrate concentration) is pleted against time. The inital slope ofthis curve is estimated (ie. = {Ps = df SV). This value of» then dopends on the values of Eq] and (Sin ‘he charge fo the reactor Many such experiments ca be used to generate many pairs of ‘and [SI data. These could be ploted asin Fig. 3.3, but the accurate determination of K, from such a plot is very dificult, Consequently, ober methods of analyzing such data have ben suggested. 3.33.1. Double-reciprocal plot (Lineweaver-Burk plot) canbe linearized in double-eciprocal frm: Equation 3.126 ay A plotof tv versus 1] ysl nar line witha slope of KV, and y-axis intercept of| Mya dopcted in Fig. 35. A double-ecirocal pa gives good estimates on Vs, But not necessarily on K,. Because the error about the reciprcal of a data point is not symmetric, the eader shouldbe cautious in applying repression analysis (last squares) 1o such plots. Data points at low substate concentrations iaflence the slope and intercept more than those at high substrate concentrations, os Emaymes Chap. 3 3.83.2, EadieHofstee plot. Equation 3.12b canbe rearranged as 2¥4-: Ke 6.14 cS] a A plot of» versus 5] resus in a Tine of slope ~Kry and Janis intercept of Vas 8 de picted in Fig. 3.6. Eadie-Hofstce plots can be subject 0 larg: errors since bth coe ates conta v, but hee i less bits on points ato [S) 3.3.3.3. Hanes-Woolf plot. Rearrangement of eq 3.12 yields {A plot of [SV versus {S] results in a Tine of slope 1/V and y-axis imercept of Kyla 8 I indicates pstve cooperativity. Figure 3.8 “Gaparey Michaelis-Menten kinetics with allosteric enzyme kistics, indicating a sig tmoidal sage of » (3) plot fr allosteric enzymes “The cooperativity coefiien can be determined by rearranging eq 3.1845, nin(S}~In ks 6.19) and by pling ln {Vq~ ¥) versus InfS] a8 depicted in ig. 3.9. 3342. Inhibited enzyme Kinetles, Cerwin compounds may bind 10 en zymes and reduce sr activity. These compounds are knowin 10 be enzyme inhib. Ea Zpme inhbiions may be lversible or reversible, levers inhibitors such a heavy Trctals (lead, cadivm. mercury, an ober) form a stale complex with enzyine andre ‘doco ensyane activity. Such enzyme inbibien may be reversed only by using chelating gent such as EDTA (ehylenediamineltracetc ci) and citrate, Reversible inhibitors tay dinsctte more easily from the enzyme alter binding Te three major classes ofr Soc.33 Entyive Kinetics o Allontrie Fero34, Compton Mie ° (s] ewes ons ennye iss serie enzyne inkbions ae competitive, noncompetitive, and yacompettve fk tons The muburate may ae or an hbitri ome cases Competve inhibi aru soba anlogrand compete wih substrate forthe scien le enzyme. Te competiivecnymsnhtion eee con be dese 5 E+SSEEES GES? 1 { (29 EI oh Slope © 9 ls at gz tog [8] 0 coat Ho Kt = Enaymes Chap. 3 Assuming rapid eqiitrium and with the definition of {11S} eu Tes] cy 62 [E)=(E}+1ES}+(E0) and y= 46S) ‘vs cam develop the following equation forthe ate of enzymatic conversion: wish ya Hp oa ve ValS) “The net effect of competitive inhibition isan increased valu of Ky and, here fore, reduced reaction ras. Competitive inhibition can be overcome by high concent tions of substrate, Figure 3.10 dserbes competitive enzyme inibtion inthe form of a ouble-reciprocal plot ‘Noncompetitive inhibitors are not subsrate analogs. Inhibitors bind on sites other than the active site and reduce enzyme afity tothe substrate. Noncompetitive enzyme inhibition ean be described as follows: E+S SESE +P With he detnition. EIS} _ (EMS) TES) ~ (ESI (Ep1=1E}+1ES]+(E0)+(ESI) and »=41ES] Gas wo can develop the following rate equation: See.33 Enzyme Kinetics 6 ) Uncampatiive wy bo k Teo none} ) Non competitive 4) Substrate Inhibition Wy poe Wy 1-0 “he 3) Yew Msi Fere310 it flab came ee om "The net effet of noncompetitive inhibition is & reduction in Vy High subse con cenisions wows not overcome noncompetitive inbibition. Other reagents need to be tuded to block binding of the inhibitor cote enzyme. In some forms of noncompetitive inhibition Vis reduced and Kis increased This cccurs if de complex. EST can form rode, ‘Uncompetitve inhibitors bind tothe ES compies only and have no afiny forthe enzymosel, The scheme for uncompetitive inhibition ts 7 Enaymes Chap. E+S

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