ENZYMES - REGULATION

REGULATION

What defines a regulatory enzyme?

In the cell, several enzymes work together in so called “pathways”, and the product from one enzyme is hence the substrate of the next. In these pathways, there are often one or more enzymes that affect the rate in a significant way – such enzymes are called : regulatory enzymes! They are special in the sense that there activity is dependent on different signals in the environment! Since the regulatory enzymes change their activity in response to different signals – the entire pathway is regulated – in response to the cells constantly changing needs!

Regulatory enzymes tend to be multi subunit enzymes.

Several types of regulation may occur in the same enzyme.

Which enzyme in a pathway is usually the regulatory enzyme?

It is usually the first one. This is a way to save energy, of course.

Enzymes may be regulated by different mechanisms, such as : substrate level regulation, allosteric regulation and covalent regulation. Describe the principles of these different mechanisms!

Substrate level regulation of enzymes leans on the previously described steady-state kinetics. That is : as long as you keep the substrate level under the limit of Vmax, you can speed up the catalytical process by adding more substrate, and slow it down by decreasing assess to the same. I think...

Allosteric regulation means that an allosteric enzyme (an enzyme with allosteric sites in addition to active sites) are regulated by so called modulators that bind to the allosteric sites of these enzymes. When the modulators bind; two things can happen : positive modulators increase the enzyme activity whereas negative modulators decrease the enzyme activity.

Covalent regulation means that the enzyme activity is altered by covalent modification of one or more amino acid residues in the enzyme! For instance phosphorylation of an amino acid may activate a “sleeping”, inactive enzyme. Another example is the introduction of an hydrophobic group, which often gives increased interaction with the cell membrane!

What is the most important type of regulation?

Probably phosphorylation. One third of the proteins in an eukaryotic cell are phosphorylated at one point or another.

What kind of enzymes catalyzes phosphorylation? What kind of enzymes remove the phosphoryl group from the enzyme?

Protein kinases are responsible for phosphorylation.

Protein phosphatases remove the phosphoryl groups.

What does phosphorylation mean in terms of chemical properties?

If phosphoryl is added to a Ser, Thr or Tyr – a charged group is introduced to an area that was only moderately polar to begin with. The phosphoryl group contains oxygens that may engage in hydrogen bonding.

Give some examples of entire proteins that may be used as modifying groups!

Ubiquintin and sumo!Thiamine pyrophosphate (TPP) is a co-enzyme facilitating the decarboxylation reactions catalyzed by both pyruvate dehydrogenase and pyruvate decarboxylate. Which property of the TPP facilitates this reaction?

The overall reaction is : pyruvate to acetaldehyde, catalyzed by pyruvate decarboxylate. This reaction requires TPP and Mg2+. TPP is a co-enzyme of pyruvate decarboxylate, and it is derived from vitamine B1. The functional part of TPP, that enables the reaction, is the thiazolium ring. See picture below :

The second carbon of the ring has an acidic H bound to it. Loss of this proton renders a carbanion which is the active agent in the following reactions. Normally, reactions that form carbanions are highly unfavorable, but in this case the positive charge on the tetravalent nitrogen next to the carbanion stabilizes the negative charge. The thiazolium ring acts as an “electron sink”, which is a very important feature in decarboxylation reactions. Electric sinks are, by definition, such groups that pulls electrons from a reactive center (in this case a carbanion) and thus stabilize an electron-deficient intermediate or transition state.

The carbanion of the TPP can perform a nucleophilic attack on the carbonyl group on the substrate. (This forms a single bond between the TPP and the substrate.)

The detailed description below is nicked from wikipedia :

1. The target bond on the substrate is broken, and its electrons are pushed towards the TPP. This creates a double bond between the substrate carbon and the TPP carbon and pushes the electrons in the N-C double bond in TPP entirely onto the nitrogen atom, reducing it from a positive to neutral form. 2. In what is essentially the reverse of step two, the electrons push back in the opposite direction forming a new bond between the substrate carbon and another atom. (In the case of the decarboxylases, this creates a new carbon-hydrogen bond. 3. In the case of transketolase, this attacks a new substrate molecule to form a new carbon-carbon bond.)In what is essentially the reverse of step one, the TPP-substrate bond is broken, reforming the TPP ylid and the substrate carbonyl.

What defines allosteric binding?

It is noncovalent!!!! It is reversible!!!!

What defines an allosteric enzyme?

They undergo conformational changes upon binding modulators that are either negative (inhibition) or positive (activation). Sometimes a modulator can be the substrate it self.

They are often rather big enzymes with several subunits!

What kind of effect do you get from a positive modulator? From a negative modulator?

Stimulation and inhibition respectively.

What is the difference between mixed or uncompetitive inhibitors and allosteric negative modulator?

Inhibitors do not necessarily induce conformational change! And the kinetic effects of the inhibitors are very distinct.

How does allosteric kinetic behavior differ from Michaelis-Menten -kinetics?

Michaelis-Menten produces a hyperbolic curve (which shows that they are NOT regulatory and that they are MONOMERIC).

Allosteric enzymes that work through cooperativity usually produce a sigmoid curve, rather than an hyperbolic one.

What does a sigmoid curve tell us about the enzyme?

That the enzyme in question is allosteric and shows positive cooperativity! It is probably homotrophic!

Define cooperativity!

A change in one subunit will be transmitted to adjacent subunits through the non-covalent bonds between them. In other words : binding of a substrate or a ligand affects the affinity for a substrate or a ligand on other binding sites in the enzyme!

In enzymes consisting of several subunits, this increased affinity will cause a rapid change in the velocity of the catalytical process until Vmax is achieved. Plotting the V0 vs [S] for a cooperative enzyme thus results in a sigmoid curve with low catalytical velocity at low substrate concentration and a fast and immediate increase in enzyme activity to Vmax when [S] is increased. Positive cooperativity implies allosteric binding – binding of the ligand at one site increases the enzyme’s affinity for another ligand at a site different from the other site Enzymes that demonstrate cooperativity are defined as allosteric. However : there are several types of allosteric interactions: (positive & negative) homotropic and heterotropic. How then, can you detect other kinds of allostery?

In heterotropic enzymes, the kinetic curves may vary! It is therefor hard to generalize regarding the kinetics in these cases!

An activator may give a more hyperbolic (M&M-kinetic) effect with decrease in K0,5 but consistent Vmax.

An activator may also increase Vmax (the efficiency, so to speak) but not the affinity (Km).

An inhibitor may give a higher K0,5 (the affinity is decreased and more substrate is required for Vmax to be reached). In short : heterotropic allosteric enzymes show different kinetic behavior because they are affected by activators, inhibitors or both!

What is the actual difference between non-competitive inhibition and allosteric inhibition?

Noncompetitive vs. allosteric inhibition: noncompetitive inhibitors bind to a site other than the active site and render the enzyme ineffective. Allosteric inhibitors do the same thing. So, how are they different? And, in what way can we apply the Michaelis-Menten equation to our understanding of allosteric inhibitors? For instance, can we quantify what happens with the presence of an allosteric inhibitor, or do we just have a qualitative understanding?
I agree that at a simple mechanistic level non-competitive and allosteric inhibition appear the same. There are several differences, however. Allosteric inhibition generally acts by switching the enzyme between two alternative states, an active form and an inactive form. It usually works by binding to a sites in a specialized subunit of a multimeric protein, and thus binds at several sites. The more inhibitor that binds, the more then can bind, and vice versa with substrate. The kinetics are thus complicated, being cooperative, and non-Michaelis Menten, and are beyond the scope of this course. So a qualitative understanding is all that is called for here. Allosteric inhibition is designed into the proteins and represents an important physiological process.
Noncompetitive inhibition is more of a catch-all for non-physiological inhibition that does not compete with substrate for substrate binding to enzyme. In that, it is defined (and named) from a negative point of view. A non-competitive inhibitor may bind to a non-substrate site on a protein and distort it to the point of non-functionality, and adding more substrate will not alleviate this inhibition. Or it may simply block a catalytic site without interfering with substrate binding, an example that is more distinct from allosteric inhibition.

How can allosteric regulation be detected in an enzyme reaction?

Homotrophic regulation through cooperativity can be readily detected because of the sigmoid curve resulting from measuring kinetics. When it comes to heterotropic allosteric regulation it´s a different story. The curves produced by these reactions do not follow any particular, predictable pattern and are often impossible to separate from mixed/un-competitive inhibition. Hence, kinetics is not useful in these cases – other, very advanced and still not very reliable methods are being used.

Competitive and non-competitive inhibitors bind reversibly. An inhibitor that binds covalently to irreversibly inactivate the enzyme is called an irreversible inhibitor or inactivator.

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