Cellular and Molecular Biology Topics                                                                

Enzyme Regulation

Allosteric Interactions

Allosteric effectors can influence the way multi-subunit enzymes function and cause their active sites to behave in a cooperative manner. In other words, binding substrate to one active site can influence how substrate binds to another site. Rather than hyperbolic saturation curves, these enzymes show sigmoidal or “S-shaped” substrate saturation curves.

These effectors can either cause positive cooperativity or negative cooperativity. In positive cooperativity, substrates facilitate binding at other active sites and are known as allosteric activators. In negative cooperativity, substrates discourage binding at other sites, and are known as allosteric inhibitors.

The nature of cooperativity can be obtained from the Hill equation.

v = Vmax [S]^n
      Ks + [S]^n

n > 1 positive cooperativity
n = 1 simple Michaelis-Menten kinetics
n < 1 negative cooperativity

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Advance Topics: Intro to Pharmacology & Toxicology

Globins

Both hemoglobin (Hb), the oxygen carrier molecule, and myoglobin (Mb), the oxygen storage molecule, belong to the class of proteins known as globins. Both contain a heme group and perform similar functions involving the reversible binding of oxygen.

       
                          Hemoglobin                                                         Myoglobin

The active site of both Mb and Hb is iron (Fe2+) at the center of the heme prostethic group. The Fe2+ iron is bound by four nitrogens of the heme group, the proximal His of one of the monomers and by oxygen when present. A distal His does not coordinate to the Fe2+ but helps select for O2 binding by forcing ligands to bind at an angle to the heme plane (the same angle of the oxygen molecule). This is the natural position for O2 binding, but creates strain for CO binding.

The O2 binding site is also shielded from water by hydrophobic amino acids. This prevents the oxidation of Fe2+ (Fe2+ + O2 Fe3+ + O2-), which forms methemoglobin, a form non-functional as an O2 carrier. Methemoglobin reductase can regenerate the Fe2+ form of Hb.

Monomeric Mb exhibits a hyperbolic saturation curve for O2 binding. This is typical of proteins with binding sites acting independent from each other. Tetrameric Hb exhibits a sigmoidal curve instead. This type of binding curve is characteristic of positive cooperative interactions between binding sites.

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Advance Topics: Biochemistry

Cooperative O2 Binding

The difference in binding between the monomeric Mb and the tetrameric Hb helps explain the theories of allosteric and cooperative behaviour of proteins. In Hb, each monomer aids the other monomers to assist in O2 binding. Cooperative binding of O2 enables Hb to deliver 1.8 times the amount of O2 to muscle and tissue under normal physiological conditions than a non-cooperative carrier. Binding of O2 to one subunit in deoxy Hb causes structural changes in the protein which are communicated to the other subunits in the protein by a conformational “switch” between the deoxy state (T or tense) and the oxy state (R or relax).

Allosteric effectors of O2 binding to Hb include pH, CO2 and 2,3-diphosphoglycerate (2,3-DPG). A decrease in pH (elevated [H+]) promotes O2 release. The oxygen binding curve is shifted to the right. This is known as the Bohr effect. Lower pH stabilizes the deoxy state.

Elevated CO2 promotes O2 release in two ways: by the carbonic anhydrase reaction or by direct binding. CO2 is a relatively insoluble gas, it must be hydrated in red cells by carbonic anhydrase:

CO2 + H2O HCO3- + H+

Hb can pick up these protons as it deoxygenates, allowing more CO2 to be hydrated (i.e. pulls the reaction to the right).

When deoxy Hb reaches the lungs (low [CO2] and lower [H+]) it releases its protons as it picks up O2. The release of H+ and low [CO2] pull the reaction catalyzed by carbonic anhydrase to the left.

CO2 binding to the N-terminus of the a-chains forms carbamates:

R-NH2 + CO2 R-NHCOO- + H+

These adducts stabilize deoxy Hb, thus shifting the saturation curve to the right.

2,3-DPG is produced in red blood cells (RBC) from the glycolytic intermediate 1,3-DPG. It is present in RBC at approximately equimolar concentrations with Hb. 2,3-DPG binding stabilizes the deoxy Hb forming salt links with lysines and histidines of the b-chains as well as the amino terminus of the b-chains. This shifts the saturation curve to the right. There is one binding site per tetramer.

The body’s short-term adaptation (1-2 days) to high altitude is to increase the levels of 2,3-DPG in RBC.

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Advance Topics: Biochemistry

Enzyme Regulation

Enzymes may be reglate by allosteric interactions or covalent modifications. A product of an enzymatic reaction can act as an allosteric inhibitor of the enzyme. The end product of the pathway controls the flux through the pathway, preventing too much product synthesis, which would be energetically wasteful.

The enzyme aspartate transcarboxylase is an allosteric enzyme that catalyzes the first step in CTP synthesis, and is regulated by allosteric interactions. ATP is an allosteric activator and CTP is an allosteric inhibitor. Other examples of allosteric regulation include fructose-2,6-phosphate (F-2,6-P) activation of phosphofructokinase (PFK) and allolactose activation of the ac operon. F-2,6-P is formed by the cell when an excess fructose-6-phosphate, the substrate for PFK, is present. Allolactose, a metabolite of lactose, activates the transcription of genes responsible for lactose metabolism in bacteria by binding to and inactivating the lac repressor.

There are two basic types of covalent control of enzyme activity: reversible and irreversible. Examples of irreversible covalent control includes activation of proenzymes, prenylation and acylation.

The key reversible covalent modification of activity is phosphorylation. Protein kinases use ATP to transfer a phosphate to a protein at a Ser, Thr or Tyr residues while protein phosphatases hydrolyze the phosphorylated protein with water. Note that if unregulated this becomes a futile cycle of ATP hydrolysis. Thus, the reactions must be tightly controlled by the cell, usually as a response to an extracellular signal.

An example of regulation by phosphorylation is the regulation of the pathways of glycogen synthesis and breakdown. Glycogen synthase synthesizes glycogen from glucose, and glycogen phosphorylase breaks down glycogen to glucose. Phosphorylation inactivates synthase but activates phosphorylase. The cell responds to the hormone epinephrine by activating protein kinase that phosphorylate both enzymes. Insulin dephosphorylates both enzymes by activating phosphatase.

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Advance Topics: Biochemistry

Topic

Enzymes

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Advance Topics: Biochemistry

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