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BIOC12Fall2012 lecture week 12 notes

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Biological Sciences
Rongmin Zhao

1 BIOC12Fall2012 Lecture Week 11: Enzymes – specificity and mechanisms of action (Chapter 13 and 14) The general property of enzymes 6 12 o Higher reaction rates: typically catalyzes the reaction 10 -10 times faster o Milder conditions: reactions occur in physiological conditions o Greater reaction specificity: the spectrum of reactant (substrates) or product is very narrow o Capacity for regulation: the catalysis rate can be changed or regulated  change in pH causes enzyme to become inactive First step of catalysis o The first step is binding between the enzyme and the substrate o Enzymes catalyze reactions by lowering activation energy o o When the substrate is bound by the enzyme, the enzyme’s energy is lowered o If the E binds to the substrate so tight then the energy is so low that the reaction does not happen o Catalysis does not occur if ES and X are equally stabilized  if X is stabilized more than ES then catalysis will occur o o EX is the transition state  frm ES to EX the activation energy needs to be overcome o If the activation energy is the same as without the enzyme (fig 1) then the reaction catalysis will not occur  i.e. havng the enzyme makes no difference o If activation energy is not decreased there is no enhancement of reaction rate ES complex adopts lower energy state 2 o o formation of the ES complex results in entropy loss o the ES complex is a more highy ordered, low entropy state for the substrate o so there is a drop in overall energy  big bump to overcome to TS o o substrates typically looses water of hydration in the formation of the ES complex o desolvation raises the energy of the ES complex, making it more reactive o this is not stable o this means that the distance between the transition state and ES will be lower  catalysis will probably occur because the small hill of activation energy will be able to be overcome o so basically when the complex of enzyme + substrate makes a more disorded molecule than each on their own, then some of the activation energy is overcome this way, therefore there is less to be overcome until the TS is reached, and so reaction progresses at a faster rate o o electrostatic destabilization of a substrate may arise from juxtaposition of like charges in the active site o if charge repulsion is relieved in the reaction, electrostatic destabilization can result in rate increase o it allows the ES complex to not be tightly bound  enzyme does not catalyze reaction if tight bind Mechanisms of Catalysis o preferential binding of transition state complex o enzymes facilitate formation of near-attack complexes o protein motions are essential to enzyme catalysis o covalent catalysis o general acid base catalysis o metal ion catalysis 3 How Tightly Transition state Analogs bind to the active site o very tight binding to the active site -20 -26 o the affinity of the enzyme for the transition state may be 10 to 10 M o can we see anything like that with stable molecules? o Transition state analogs (TSAs) are stable molecules that are chemically and structureally similar to the transition state o Proline racemase is a good example o o Pyrrole-2-carboxylate and Δ-1-pyrroline-2-carboxylate mimic the planar transition state of the reaction o The C in the transition state is planar from its original tetrahedral structure o The proton then comes back and attacks from either side resulting in either L or D proline o There are 2 other chemicals that can be used to mimic the planar transition state  in these mimics they noticed that binding was very tight o o purine riboside inhibits adenosine deaminase o the hydrated form is an analog of the transition state of the reaction o If the different chemical is used, then you can find the K  which once again shows that the binding is very tight o You can treat it as an inhibitor because the rate is lowered because it binds so tightly o You can treat the Km of the TS as a Ki  the way we define Ki and Km are the same (we use EQ hypothesis) o Transition state analog is very stable and mimics the actual transition stae  we can study analogs to see how tightly bound the actual transition states really are Near-attack complexes 4 o X ray crystal structures studies and computer modeling have shown that the reacting atms and catalytic groups are precisely positioned for their roles o Such preorganization selects substrate conformations in which the reacting atoms are n van der Waals contact and at an angle resembling he bond to be formed in the TS o Bruice has termed such arrangement near-attach conformations (NACs) o o NACs are characterized as having reacting atoms within 3.2A and an approeach angle of 15 degrees of the bonding angle in the transition state o o in an enzyme actie site, the NAC forms more readily than in the uncatalyzed reaction o the energy separation between the nAC and the transition state is approximately the same in the presence and absence of enzyme o example: active site of liver alcohol dehydrogenase – a near attack complex o o the enzyme binds the substrate (benzyl alcohol) n such a position that it is in very close contact to NAD+  deprotonates alcohol and breks it down into ketones and aldehydes Protein Motions are Essential to Enzyme Catalysis 5 o proteins are constantly moving  bonds vibrate, side chains bend and rotate, backbone loops, wiggle and sway and whole domains move as a unit o enymes depend on such motions to provoke and direct catalytic events o protein motions support catalysis in several ways o active site conformations can: o assist substrate binding, bring catalysitc groups into position, induce formation of NACs, assisnt in bond making and bond breaking, facilitate conversion of substrate to product o i.e. conformational changes in enzyme facilitate the reaction o example: human cyclophilin A is aprolyl isomerase which catalyzes the interconversion between trans and cis conformation of proline in peptides and proteins o o the active site of cyclophilin with a bound peptide containing proline in cis and trans conformations  pushes proline to change conformation o motion by active site residues promote catalysis in cyclophilin o so both the substrate and the enzyme change conformation during catalysis reactions Covalent catalysis o some enzymes derive much of their rate acceleration from formation of covalent bonds between enzyme and substrate o the side chains of aa in proteins offer a variety of nucleophilic centers for catalysis o these groups readily attack electrophilic centers of substrates, forming covalent enzyme-substrate complexes o the covalent intermediate can be attacked in a second s
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