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Student Questions and Answers October 15, 2002 Q 1. Water molecules interfere with enzyme-substrate interactions, so in active sites of enzymes water is missing. How is this water-missing-environment provided? Enzymes work within cells, in a water-environment (cytoplasm). Answer: FK: In most cases one or a few molecules of water are found in the empty active site, which have to be displaced by the substrate(s) upon binding. In some cases water is completely restricted from the active site area by apolar side chains. In any case, as illustrated by the presented figures, when the substrates interact with the enzyme surface there is no layer of hydrate between them! Q 2. Collision of substrate and enzyme molecules is a matter of chance. Are enzymes evenly distributed within the cell or do they accumulate on certain locations to facilitate these collisions? Answer: FK: Enzymes (and other macromolecular components) usually are not evenly distributed. This is due to a) compartmentation, b) at least loose interaction with other cellular components (including membranes and cytoskeleton), and c) limited diffusion under in vivo conditions. [As already illustrated by AG, the contents of cells and organelles represent a rather “thick soup”. This phenomenon has been called “macromolecular crowding”, since the total “concentration” of different macromolecules and their assemblies ranges from 20 up to 40% (w/v). Major consequences: due to their size only about 10% of the total volume is really accessible for macromolecules; diffusion coefficients of proteins are only 5 – 20% of those observed in water; activity coefficients can be as high as 100, i.e. the apparent concentrations (activities) which are relevant for any reaction can be significantly higher than the true concentration; this also generally leads to much higher association constants. Taking all together there is a good chance of productive collision. Q 3. If substrate/enzyme impact is a stochastic process, how can reactions proceed fast enough? Are the concentrations of both partners high enough that sufficient reaction encounters will occur any way? Answer: FK: see above Q 4. Proteins stabilize the transition state, thus preventing it falling back to the reactants. On the other hand it can transform to the products. Why? Answer: FK: Stabilisation does not mean frozen, it just says that at ordinary temperatures reactants have a fair chance to get to this (and through) stage. Think of the simple picture of pulling a car on top of a hill. From there it can roll back or to the other side, you would not expect it to stay there! (but obviously the lower the hill, the more often you will manage to get it there!) Q 5. In aqueous solution ionic substrates will be hydrated. So how is it achieved that the substrate gets free (of its hydrate shell) to bind to an enzyme? (asked twice) Answer: FK: Same story as with protein folding. The hydrate shell has to be stripped off, which costs some energy, since weak bonds are broken. This energy obviously has to come from even more interactions between substrate and the active site. Q 6. Where does the activation energy in an enzyme come from? Is this achieved only by weak bonds or are there other forces involved? (asked twice, already discussed) Answer: FK: Many factors will (could) contribute: The most important should be a) proximity: enzyme catalysed reactions virtually are intramolecular reactions, since the reactants are fixed as close neighbours, whereas in free solution intermolecular reactions take place; (an example: the uncatalysed reaction of a carboxylic acid and an ester with formation of an anhydride is more than 100-times faster if it proceeds intramolecular rather than intermolecular, see below) O COOR O COOO b) c) d) e) f) g) h) ‡ O CH3 - C - OR + O CH3 - C - O- orientation factor according to induced fit hypothesis some strain is placed on the substrate intrinsic binding energy complementarity to and thus stabilisation of the transition state protein mobility entropy effects reaction pathway may be different from that in solution ’ individual substeps each with relatively low activation energy Q 7. How does enzyme catalysis work with hydrophilic substrates which don’t have a hydrophobic tail? Answer: FK: question not clear Q 8. Within cells all enzymes and substrates “swim” in an aqueous solution; how can there be no water at the active site of an enzyme? How can the water be pushed out (when there are polar amino acids)? (asked four times) Answer: FK: see above, Q 5. You have to take into consideration, that (binding) equilibria represent a dynamic situation. So bonds, especially those involving water, are constantly broken and rebuilt, so a slightly favourable interaction will establish without much effort. Q 9. Does the enzyme favour (stabilize?) the transition state or the substrate? Answer: FK: It stabilises the transition state, but of course it also must show sufficient affinity for the substrate, otherwise it would never bind. Q 10. Has the transition state been studied in vitro, i.e. is the structure of the transition state just hypothetical or do we have experimental data? Answer: (already mentioned in the lecture) FK: various transition state analogs have been synthesised which indeed act as very potent competitive inhibitors (i.e. they bind with higher affinity than the substrate). Q 11. Could a similar, but wrong substrate inactivate an enzyme (blocking it if strong enough to withstand thermal motion)? If it is so, how may this mismatching be removed? Answer: FK: Exactly what transition state analogs do. But as long as no covalent interaction takes place this binding will be reversible. Q 12. To what extent do enzymes lower the activation-energy of a substrate? Answer: FK: Typical rate enhancements for enzymes over the corresponding uncatalysed reaction are between 108 and 1016 (!). Since k ~ e-∆Gact/RT , kcat/kuncat = e-∆∆Gact/RT, and thus this enhancemant corresponds to reductions of 40 to 80 kJ (at 300K). Q 13. How can the enzyme lower the activation energy of a reaction (to reach the transition state) when, in fact, as you mentioned, the substrate turns into the transition state by itself? Does the enzyme catch the substrate a little bit earlier? Is the transition state with an enzyme at a lower energy level? Answer: (answer given in the lecture) FK: The transition state is stabilised (at a lower energy state) and therefore its formation is more likely. From the above relationship even a reduction of ~2.5 kcal (corresponding to one single extra hydrogen bond) will lead to a 100-fold increased rate of transition state formation. Q 14. How many reactions can one enzyme (molecule) catalyse until it is destroyed? Answer: FK: With the exception of very reactive substrates which might undergo sidereactions within or outside the active site (e.g. radicals, hydrogen peroxide, oxygen), an enzyme theoretically could proceed indefinitely. Actually all proteins are subject to various covalent modifications (ageing), and since there is no repair system for proteins they are recycled sooner or later. Q 15. As to the secondary structures of enzymes: are α-helices or β-sheets preferred? Is there any difference or connection between structure and function? Answer: FK: Unclear what you really mean. Secondary structure is predicted by the respective amino acid sequence. Thermodynamically β-strands tend to be slightly more stable than helices, but this is far than outweighed by kinetic factors. If you mean the relative abundance: the average polypeptide contains 25% α-helices, a few % of 310helices and 20 to 25% β-strands. Generally smaller proteins are richer in helices, larger ones prefer β-sheets or αβ-super-secondary elements. There is no correspondence between structure and function in that sense that some kind of catalysed reaction requires either of the two types of secondary structure. Usually active sites are formed in clefts between domains or super-secondary structures, so different structural elements contribute to the catalytically essential residues. Q 16. Is there any way that induced fit happens with other molecules than specifically with the substrate? (a few weak bonds are enough …) Answer: FK: Generally it´s more than a few, but if some molecule is related closely enough than it effectively mimics the substrate and, of course, also induced fit will occur. Q 17. The formation of the state of induced fit has to be endergonic (otherwise it would happen already in the absence of substrate). Where does this energy go after/during the reaction? Is it part of the whole reaction kinetics? Answer: FK: ? Q 18. Can 2 enzymes bind at the same time to the same substrate? Answer: FK: For steric reasons this could (and in fact does) only happen with a macromolecular substrate. Q 19. How many substrates can an enyme bind? (asked twice) Answer: FK: a) If you mean different kinds of substrates this can be up to 3 (terreactant reactions). But even with only 2 substrates, they do not necessarily come together at the active site: numerous enzymes reveal a “ping-pong” mechanism. The enzyme reacts with the first substrate and releases it, itself being transformed to some modified intermediate enzyme species. This species then (and only then) can bind the second sustrate and reaction takes place. Upon release of the second product the resting form of the enzyme is recovered. b) At (very) high substrate concentrations many enzymes will bind 2 or even more substrate molecules at or close to the active site, thus preventing turnover (substrate inhibition). Q 20. Is the enzyme-substrate specificity only determined by their shape? Answer: FK: Both, shapes and (distribution of) charges determine how good the active site of an enzyme and a potential ligand fit to each other. Q 21. How does the enzyme change its shape, so that the substrate fits? To which degree is the change possible? Answer: FK: Upon inspection of protein structures you will quite often detect whole hydrogen-bonding or charge-dipole interaction networks. In some cases local disturbances can lead to a breakdown of the entire network, thus destabilising the respective conformation. Usually the loss of interaction energy is easily matched by the formation of some alternative network, now including the bound ligand. Again folding (or partial refolding in this case) is a highly co-operative process, quickly affecting large parts of the protein. In most cases the induced conformational change can be discribed as a rearrangement of domains or parts of domains relative to each other. Q 22. The enzyme binds to the transition state of a substrate. Could it be the energy of the transition state provides the energy for induced fit and conformational changes to push the reaction? Answer: FK: The transition state effectively is not available in solution, so the enzyme can´t bind it. So induced fit reflects energy “freed” upon enzyme-substrate binding, but this induced change of conformation facilitates transition state formation. Q 23. An enzyme binds substrates which then change to the transition state, but why does the enzyme not bind the transition state? Or is this too slow for biochemical reactions (not enough collisions for enough transition states)? Answer: FK: see above Q 24. Is the “lock and key” theory only an “old” theory, or do enzymes exist which don’t change their conformation upon substrate binding (induced fit)? Answer: FK: Hard to say. Many examples of induced fit are beyond doubt, since major rearrangements are seen in structures with and without substrate (or dead end competitive inhibitor). But there are also examples where you only see minor dislocations of a very few atoms upon substrate binding, what you hardly would call induced fit. In most cases the respective structures are not yet available at atomic resolution. Q 25. Is every enzyme directly involved in the catalyzed reaction (like lysozyme donating hydrogens)? Answer: FK: If you include cofactors and prosthetic groups the answer most likely is yes. Q 26. In what way can reactions with ∆G<0 be coupled with reactions with ∆G>0? Answer: FK: question too early, will be discussed in much detail Q 27. Are there any reactions in the major metabolic pathways that occur spontaneously (without needing enzymes)? Answer: FK: Hardly any, with the exception of unwanted ones like hydrolysis of ATP. Even very simple spontaneously occuring reactions like CO2 + H2O « HCO3- + H+ are catalysed (carboanhydrase). Q 28. Are there 1-step reactions which require more than one enzyme to occur? Answer: FK: Matter of definition. As mentioned above the success of enzymatic catalysis to some degree is based on specific reaction paths, which may include several substeps each catalysed by its own enzyme (or active site in a multifunctional enzyme protein). Q 29. Can a substrate molecule (already bound to the enzyme) be set free again without being altered or conversed to product, and if so, does it require energy? Answer: FK: Binding (by definition) is reversible, so a bound ligand can get off again. Binding, however, is energetically favourable (see above), so dissociation requires energy. Q 30. Would it be possible to mutate an enzyme so that it would prefer another substrate very closely related to the original substrate (i.e. glucose instead of galactose)? Answer: FK: Yes, in fact one of the major topics of modern protein engineering. Q 31. Amino acid charge: how do proline groups (the NH/NH2) dissociate? Answer: FK: The imino is group is an almost one order of magnitude stronger base than the amino grops of all other amino acids (pK 10.6 as compared to 8.4 to 9.9). After peptide bond formation protonation of peptide-nitrogens no longer plays any role. Q 32. You said that hydrogen bonding is a straight line in its strongest form. So, is the antiparallel form of β-pleated sheet more stable than the parallel form (hydrogen bonds are not in a straight line)? Answer: FK: Antiparallel beta sheets are thought to be intrinsically more stable than parallel sheets due to the more optimal orientation of the interstrand hydrogen bonds and also because there is no resulting dipole moment. Accordingly, they can tolerate bends and other distortions more easily than parallel sheets. On the other hand, a survey of hydrogen bonds in protein 3D-structures revealed no significant difference in the linearity of the hydrogen bonds in parallel and antiparallel sheets. So the answer is not a clear yes.