1、Absorption Spectroscopy/Protein Function,Topic 4 Part 2 Biophysics,Chemical Kinetics Zero Order,Rate independent of concentrations -dC/dt = k C(t) = C0 kt,Reaction of nitrite with deoxyhemoglobin,Chemical Kinetics First Order,-dCA/dt = kCA , CA = CA0 e-kt t1/2 = ln(2)/k; t = 1/k = lifetime,NO bindin
2、g to Hb,Chemical Kinetics Second Order,-dCA/dt = -dCB/dt = kCACBMake one species in excess so get pseudofirst order kinetics, kobs = kCB so CA = CA0 exp(-kobst),Hemoglobin,Cooperative Binding of Oxygen Linked to quaternary structure Explained by MWC Model,On the Nature of Allosteric Transitions: A P
3、lausible Model,Jacques Monod, Jeffries Wyman, Jean-Pierre Changux J. Mol. Biol. 1965,“Molecular Amplifiers”,ATCase,The goal is control want a switch. “Indirect interactions between distinct specific binding sites (allosteric effects)”,Definitions and Generalizations,Homotrophic effects identical lig
4、ands (eg. for Hb: O2, CO, NO) Heterotrophic effects different ligands (eg. for Hb: DPG, IHP, Cl-, NO as SNO, NEM) Most allosteric proteins are oligomers (several subunits or protomers) Allosteric changes often involve quaternary stucture Heterotrophic - positive or negative, Homotrophic only positiv
5、e (exception of Hg reductase?),Model in English,Allosteric proteins are oligomers where the protomers are arranged symmetrically There is one and only one identical ligand-binding site on each protomer Tertiary structure of protomers affected by quaternary structure There are two quaternary states (
6、R and T) which dictate ligand affinities on all protomers Transitions between states preserve symmetry,Model in Math,T0 = L R0, , L is the allosteric constant, (Big L = Big allostery) Only also define,c defines relative affinities of quaternary states and a defines absolute affinity of one When L is
7、 small,Compare Hill Equation,vs,Q is a constant, n is the number of ligand sites, n is Hill coefficient,Hb, L, c ,Hb Sigmoidal,See satsimple.mw and sat.mw,Heterotrophic effectors,Affect L Activators decrease L (push to R) and Inhibitors increase it,See sat.mw,Hb microstate predictions (vs sequential
8、),Unlike in sequential model, no R2 or T2 see states.mw,ATCase and inhibitor,At low concentrations of substrate, low concentrations of analogue activate (by promoting R-state) upper curve Desensitized enzyme (quaternary interactions suppressed) shows no increase in activity by analoque (maleate) Gen
9、erally, desensitized enzymes lose cooperativity. Hb dimers are R-state like and like Mb. Homotrophic ligands promote tetramer stabilization (hard to dissociate oxyHb), as predicted,Activators can decrease cooperativity,Fig 6a is theoretical (see yf.mw ) Fig 6b and c show activations in real systems,
10、Confirmations of MWC ATCase,Model predicts fraction in R-state fraction ligand bound. Schachman lab (1966) shows this using sedimentation to examine quaternary state (size) and spectroscopy for ligation. They also showed (like Gerhart lab) low concentration of inhibitor activate ATCase,Confirmations
11、 - Hb,MWCs prediction of concomitant changes in tertiary structure in protomers with known symmetry of tetramer confirmed by more refined X-ray structures. Perutz provides mechanism of allosteric transitions Szabo and Karplus show quantitative agreement of MWC/perutz model with equilibrium data (Eg Lc4 constant after all salt bridges broken). Equilibrium oxygen binding to Hb trapped in T-state crystal non-cooperative (Eaton lab). CO rebinding following photolysis of HbCO (R-state) much faster than CO binding to Hb (T-state) Gibson.,
