Download Student Questions and Answers October 15, 2002

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.