In our notes, in non-competitive inhibition, the substrate is still able to bind to the enzyme, but then the enzyme can't do anything with it. In Purves, it says that the presence of the non-competitive inhibitor in its own site on the enzyme distorts the shape of the enzyme and therefore makes it difficult for the substrate to bind to the enzme. Which one is it? Is it a combination of both? Yes, both. Purves does not offer the explanation I prefer, and vice-versa. I like to reserve the distortion case for allosteric inhibition, which is yet another situation (more complex).
Why does Km stay constant in non-competitive inihibition? The non-competitive inhibitor does not bind to the substrate-binding site. Those enzyme molecules that are still active are responding to substrate normally, according to the intrinsic Km of the enzyme.
It seems to me that non-competitive inhibitors must bind irreversibly to
the enzyme in order to exert their effects, and that substrate binding could be
affected. What is wrong with this picture?
Irreversibility has nothing to do with it. If a particualr enzyme molecule is
(reversibly) binding a non-competitive inhibitor (NCI) , then while it is doing
so, it will be inactive and will not contribute to the formation of product,
not contribute to the velocity of the reaction. So the velocity of the
reaction will be less in that test tube for that moment because of the presence
of the NCI on that molecule at that moment. The next moment, the NCI will have
become disassociated from its enzyme
target, but another NCI will have become associated with another enzyme
molecule, thus taking it out of action. So statistically, summed over all
enzyme molecules and all the time going by, there are effectively less active
enzyme molecules in a test tube in the presence of this dynamically binding
NCI. The presence of substarte has no effect on this state of affairs, as it
binds to a different site on the enzyme whether the enzyme is active or not (by
definition for a NCI).
What does the picture 6.19 in Purves have to do with non-competitive
inhibition?
Non-competitive inhibition is defined as inhibition that is not affected by
variations in the concentration of substrate. The example I gave in lecture was
an inhibitor that prevented the k3 reaction (S -> P) without affecting k1 or
k2.
Such inhibition could be achieved by blocking an amino acid residue at the
active
site (but not at the substrate binding site of the active site) or by binding
elsewhere, deforming the enzyme so as to inhibit the k3 reaction but not
affecting its ability to biond substrate, as in the Purves example. I avoided
this example, as it overlaps with a simple description of allosteric inhibition.
Non-competetive inhibition can't be drowned out by adding more S
but can be by adding a lot of E, correct?
More S does not help restore acitivtyot an enzyme inhibited by a
non-competitive inhibitor. Addng more E always helps (increases the rate of
the reaction), no matter what the inhibiton, as long as the enzyme is not
saturated with inhibitor.
Can I say a non-competitive inhibitor (NCI) binds to the enzyme not only at a
different site, but with a much better binding ability than the substrate? So
much better that the binding of NCI to enzyme is almost "permanent" compared to
substrate binding. That's why the enzyme bound to NCI is "permanently" removed
from the game (they are dead). No matter how much substrate we put in, the
small amont of NCI (such as poison) is so potent that it binds the enzyme with
extreme ability.
No. There is no relationship between the binding affinity of a non-competitive
inhibitor (NCI) and that ofthe substrate. In most cases the binding of the NCI
by the enzyme is not "permanent" but rather the enzyme is in equilibrium
between the bound and free state. The lecture diagram showing some enzyme
molecules with the NCI bound is meant to represent only a snapshot of the
situation at a given moment in time,or an average of many such snapshots. In
reality the NCI is continually
binding to the enzyme and dissociating from it. But for the period of time the
NCI is bound (which can be considerable), the enzyme cannot function.
You said that a non-competitive inhibitor
does not change the active site of the enzyme, meaning that
the substrate still binds but it can't do anything. In the
picture link from Purves (figure 19.C), the non-competitive
inhibitor changes the enzyme's conformation so that it can't
bind the substrate. Does the substrate bind or not?
Purves says the non-competitive inhibitor affects the active
site, but not the substrate binding site (p.127, last paragraph).
The picture from the publisher's Web site differs from that in the current
Purves (4th Ed.) text Fig. 6.19C. I believe the text paints a less confusing
picture and have removed the linked picture. The active site would comprise
the the substrate binding site plus those elements that facilitate the
reaction, that help catayze the reaction, that produce a transition
state.
The catalytic activity is inhibited, but substrate binding per se
is not. The important thing is that substrates do not compete with
a noncompetitive inhibitor, and that non-competitive inhibitors do not
resemble substrates.
Why can't non-competitive inhibitors be 'swamped
out' by excess substrate?
Because they do no compete with substrate for binding. They are essentially
oblivious to the substrate concentration.