Mastering Biology: Section 8.5 - Regulation of Enzyme Activity

What is allosteric regulation?

Allosteric regulation is the term used to describe any case in which a protein's function at one site is affected by the binding of a regulatory molecule to a separate site. It may result in either inhibition or stimulation of an enzyme's activity. The molecules that naturally regulate enzyme activity in a cell behave something like reversible noncompetitive inhibitors. These regulatory molecules change an enzyme's shape and the functioning of its active site by binding to a site elsewhere on the molecule via non-covalent interactions.

Although allosteric regulation is probably quite widespread, how come relatively few of the many known metabolic enzymes have been shown to be regulated in this way?

Allosteric regulatory molecules are hard to characterize, in part because they tend to bind the enzyme at low affinity and are therefore hard to isolate. Recently, however, pharmaceutical companies have turned their attention to allosteric regulators. These molecules are attractive drug candidates for enzyme regulation because they exhibit high specificity for particular enzymes than do inhibitors that bind to the active site. (An active site may be similar to the active site in another, related enzyme, whereas allosteric regulatory sites appear to be quite distinct between enzymes. The more precise we can get, the better targeted therapies there will be, which will result in better medicine for me in the future. I might also be able to tap into these drugs in order to learn more, do more, be better, etc...

How does the cell manage its metabolic activity?

Chemical chaos would result if all of a cell's metabolic pathways were operating simultaneously. Intrinsic to life's processes is a cell's ability to tightly regulate its metabolic pathways by controlling when and where its various enzymes or, as we discuss here, by regulating the activity of enzymes once they are made.

Figure 8.2 - Inquiry: Are there allosteric inhibitors of caspase enzymes?

Experiment: Screen 8,000 compounds for their ability to bind to a possible allosteric binding site in caspase 1 and inhibit the enzyme's activity. Each compound was designed to form a disulfide bond with a cysteine near the the site in order to stabilize the low-affinity interaction that is expected of an allosteric inhibitor. Since caspases are known to exist in both active and inactive forms, the researchers hypothesized that this linkage might lock the enzyme in inactive form. Results: Fourteen compounds were identified that could bind to the proposed allosteric site of caspase 1 and block enzymatic activity. The enzyme's shape when one such inhibitor was bound resembled the inactive caspase 1 more than the active form. (Thats a good thing!.. right?) Conclusion: That particular inhibitory compound apparently locks the enzyme in its inactive form, as expected for a true allosteric regulator. The data therefore support the existence of an enzyme allosteric inhibitory site on caspase 1 that an be used to control enzymatic activity.

How is cooperativity related to hemoglobin, the vertebrate oxygen transport protein?

Hemoglobin is made up of four subunits, each of which as an oxygen-binding site. The binding of an oxygen molecule to one binding site increases the affinity for oxygen of the remaining binding sites. (seems like allosteric regulation to me). Where oxygen is at high levels, such as in the lungs or gills, hemoglobins affinity for oxygen increases as more binding sites are filled. In oxygen-deprived tissues, however, the release of each oxygen molecule decreases the oxygen affinity of the other binding sites, resulting in the release of oxygen where it is most needed. (A double edged sword?) How can I come up with a technology that when an oxygen molecule releases from the hemoglobin enzyme, the oxygen affinity does not decrease in other enzymes? How do enzymes, such as hemoglobin communicate with each other almost instantaneously?

Figure 8.20 - What if? As a control, the researchers broke the disulfide linage between one of the inhibitors and the caspase. Assuming that the experimental solution contains no other inhibitors, how would you expect the caspase 1 activity to be affected?

I expect the caspase 1 activity to remain in active form, and continue to bind to its specific substrate. [Fig 8.20] Because the affinity of the caspase for the inhibitor is very low (as expected of an allosterically inhibited enzyme), the inhibitor is likely to diffuse away. Because no additional source of the inhibitory compound is present, and the concentration of the inhibitor is very low, the inhibitor is unlikely to bind again to the allosteric binding site once the covalent linkage is broken. Therefore, the activity of the enzyme would most likely be normal. (In fact, this is what the researchers observed when they broke the disulfide linkage. Haha, I got the answer right, but not when the correct reason. I believe I can call this a false positive on my end.

Imagine you are a pharmacological researcher who wants to design a drug that inhibits a particular enzyme. Upon reading the scientific literature, you find that the enzyme's active site is similar to that of several other enzymes. What might be a good approach in developing your inhibitor drug?

I would try to find an inhibitor drug that works as an allosteric regulator, because these molecules exhibit high specificity for particular enzymes, relative to other inhibitors. An inhibitor that binds to the active site of the enzyme you want to inhibit could also bind to and block the enzymes with similar structures, causing significant side effects (god damn this is why drugs have side effects!). For this reason, you would be better off choosing to screen chemical compounds that bind allosterically to the enzyme in question, because allosteric regulatory sites are less likely to share similarity with other enzymes.

What happens if ATP production lags behind its use?

If ATP production lags behind its use, ADP accumulates and activates the enzymes that speed up catabolism, producing more ATP.

What is cooperativity?

In another kind of allosteric activation, a substrate molecule binding to one active site in a multisubunit enzyme triggers a shape change in all the subunits, thereby increasing catalytic activity at the other active sites. Called cooperativity, this mechanism amplifies the response of enzymes to substrates: One substrate molecule primes an enzyme to act on additional substrate molecules more readily. Cooperativity is considered "allosteric" regulation because binding of the substrate to one active site affects catalysis in another active site.

What happens in the simplest kind of allosteric regulation?

In the simplest kind of allosteric regulation, an activating or inhibiting regulatory molecule binds to a regulatory site (sometimes called an allosteric site), often where subunits join. The binding of an activator to a regulatory site stabilizes the shape that has functional active sites, whereas the binding of an inhibitor stabilizes the inactive form of the enzyme. The subunits of an allosteric enzyme fit together in such a way that a shape change in one subunit is transmitted to all others. Through this interaction of subunits, a single activator or inhibitor molecule that binds to one regulatory site will affect the active sites of all subunits.

What is cellular respiration?

It is the major catabolic pathway that breaks down organic molecules, releasing energy for the crucial processes of life. In eukaryotic cells, the enzymes for cellular respiration reside in specific locations within mitochondria.

How can I see so far?

It's because I stand on the shoulder of giants. (All the scientists before me).

What are the structures of enzymes that are known to be allosterically regulated like?

Most enzymes known to be allosterically regulated are constructed from two or more subunits, each composed of a polypeptide chain with its own active site. The entire complex oscillates between two different shapes, one catalytically active and the other inactive.

How do an activator and an inhibitor have different effects on an allosterically related enzyme?

The activator binds in such a way that it stabilizes the active form of an enzyme, whereas the inhibitor stabilizes the inactive form.

How do the fluctuating concentrations of regulators cause a sophisticated pattern of response in the activity of cellular enzymes?

The products of ATP hydrolysis (ADP and P), for example, play a complex role in balancing the flow of traffic between anabolic (buildup) and catabolic (breakdown) pathways by their effects on key enzymes. ATP binds to several catabolic enzymes allosterically, lowering their affinity for a substrate and thus inhibiting their activity. ADP, however functions as an activator of the same enzymes. This is logical because catabolism functions in regenerating ATP.

What happens if ATP production exceeds demand?

Then catabolism slows down as ATP molecules accumulate and bind to the same enzymes, inhibiting them (from producing more ATP!). ATP, ADP, and other related molecules also affect key enzymes in anabolic pathways. In this way, allosteric enzymes control the rates of important reactions in both sorts of metabolic pathways.

What are caspases?

They are protein digesting enzymes that play an active role in inflammation and cell death. By specifically regulating these enzymes, we may be able to better manage inappropriate inflammatory responses, such as those commonly seen in vascular and neurodegenerative diseases.

What is feedback inhibition?

When ATP allosterically inhibits an enzyme in an ATP generating pathway, the result is feedback inhibition, a mode of metabolic control. In feedback inhibition, a metabolic pathway is switched off by the inhibitory binding of its end product to an enzyme that acts early in the pathway. Certain cells use the 5 step pathway to synthesize the amino acid isoleucine from threonine, another amino acid. As isoleucine accumulates, it slows down its own synthesis by allosterically inhibiting the enzyme for the first step of the pathway. Feedback inhibition thereby prevents the cell from wasting chemical resources by making more isoleucine than is necessary. The only reason why I can see so far is because I stand on the shoulders of giants. (Wow amazing, such complexity)