Structural Biochemistry/Enzyme Catalytic Mechanism/Regulation
Proteins are regulated through a variety of different ways; some are controlled allosterically, some use different forms of enzymes that catalyze the same reaction. There are enzymes that are regulated through reversible, or irreversible covalent modification, other enzymes use proteolytic activation, with zymogens that are inactive until cleaved, and finally, some enzymes are regulated by the control of the amount of enzyme present.
Strategies of Regulation: - Allosteric Control - Multiple forms of the same enzyme - Reversible covalent modification - Proteolytic activation - Controlling the amount of enzyme present
Allosteric proteins have two different sites, regulatory and functional. As the name implies, regulatory sites are used by the protein to regulate protein function. This is done by having small signal proteins come and attach themselves to them, and send various signals to control their activity. The functional sites are sites that proteins use to perform its function. Because they contain more than one different site, these proteins often show cooperativity because one active site affects other nearby active sites.
One model of cooperativity is the concerted model, also known as the MWC model. It proposes that an enzyme can only exist in the tense (T) or relaxed (R) state but not both. They either exist completely in T state or completely in R state. When a substrate binds to one monomer of the enzyme, it shifts the equilibrium between the two states. For example, hemoglobin is a tetramer. When oxygen binds to one monomer, the equilibrium shifts from the T state to the R state. The R state favors oxygen binding whereas the T state is stabilized by a different component described later. When an oxygen is bound, the equilibrium of the other monomers also shift from the T state to the R state. As more oxygen is bound to hemoglobin, the affinity for oxygen increases.
The second model of cooperativity is the sequential model. In this model, an enzyme is either in the T or R state. However, in this model, binding of a substrate to an enzyme causes a conformation shift that causes the other monomers in the enzyme to favor binding of the substrate. In hemoglobin, binding of oxygen to a monomer causes the proximal histidine to move closer to the porphyrin ring in the heme group causing one dimer to shift 15 degrees. This conformational change causes the other monomer's affinity for oxygen to increase, thus favoring the T state.
An example of allosteric control is with the enzyme aspartate transcarbamoylase or ATCase. This enzyme catalyzes a reaction that will produce cytidine triphosphate, or CTP. To control the amount of product formed, CTP will inhibit its own formation by inhibiting the catalyst, ATCase, in a process called "feedback inhibition". It does this by making the unbound form, or the T state of the catalyst ATCase more stable. This results in shifting the equilibrium towards the unbound state, and thus lowing the affinity of ATCase to its substrate. This is one way proteins ensure they waste no energy producing excess product.
CTP is not the only NMP molecule that has an effect on ATCase, ATP also has its own effect. Whereas CTP stabilizes the T state ATP stabilizes the R state of ATCase. Stabilizing the R state of ATCase allows the substrate to bind easier and in turn the reaction rate is increased.
ATP as well as CTP are referred to as "heterotropic effects" on a allosteric enzyme such as ATCase. ATP is an allosteric activator of aspartate transcarbamolyase because it stabilizes the R-state of ATCase, effecting neighboring subunits by making it easier for substrate to bind.The increase of the concentration of ATP has two potential explanations. First being, at high concentrations of ATP signals a high concentration of purine and pyrimidine. second, a high concentration of ATP conveys that a source of energy is available for mRNA synthesis and DNA replication follow by the synthesis of pyrimidines needed for these processes.
These can also be called isoenzymes. Isozymes are homologous enzymes that have the same function, but are composed of different amino acid sequences, have slightly different structures, and or respond to different regulatory molecules or kinetic parameters such as kM and Vmax. Isozymes allow for "fine tuning" of the enzymes, resulting in enzymes that have the same function, but work in different environments, or respond to different signals. Isoenzymes may be the result of mutations that are preserved in the genome or a result of convergent evolution.
An example of an isozyme is lactate dehydrogenase, or LDH. This enzyme catalyzes a step in anaerobic glucose metabolism and synthesis. People have two forms of this enzyme, the H and the M, existing in the heart and muscle respectively. These two isozymes differ in their affinity for the substrates and the levels of inhibitors needed to inhibit them. This makes sense because of the highly different environments that exist in the heart and the muscle; the heart is highly aerobic while the muscles are anaerobic. This allows for the same function to be preformed by two "different" enzymes in two different environments.
Since isoenzymes have different structures, they may be separated and identified techniques such as gel electrophoresis.
Reversible Covalent Modification
Enzymatic activity can be modified by attaching a modifying group, such as phosphorous, through a covalent bond. The new group then changes the enzyme's reactivity, size, charge, etc.
A good example of this is phosphorylation, catalyzed by protein kinases. Phosporylation is used in many different cells in many of life's organisms because it has many good attributes making it a good tool. Of these good attributes include: ~It adds two negative charges to the protein ~Forms 2-3 extra Hydrogen bonds ~Irreversible, due to the amount of energy required to phosphorylate ~has a varying speed that can be changed due to specific needs ~can cause a cascade effect, resulting in an amplified result
Protein kinases are the catalytic enzymes that phosphorylate a protein. They use ATP as the source of phosphoryl groups as well as energy. There are dedicated protein kinases that only phosphorylate a specific protein. They recognize a specific consensus sequence, which usually includes a serine or threonine residue. Other protein kinases are multifunctional protein kinases which can phosphorylate many different proteins.
Protein Kinase A (PKA) is an example of an enzyme that is regulated by reversible covalent modification and allosteric control. The holoenzyme form of PKA forms an inactive R2C2 complex comprised of two regulatory subunits and two catalytic subunits. The two regulatory subunits contain pseudosubstrate sequences that are bound to the active sites of the two catalytic subunits, inhibiting its function. PKA is activated when four cyclic adenosine monophosphate (cAMP) molecules bind to the two regulatory subunits, removing them from the catalytic active sites, thus freeing and activating the catalytic subunits. The protein kinase A is now free to carry on its function and phosphorylate other proteins.
Enzymes can also be controlled by preventing them from functioning until a given time. For example, many enzymes are controlled by hydrolysis of certain bonds, making an inactive enzyme active. The inactive precursors are called zymogens; enzymes such as chymotrypsin, trypsin, and pepsin show this trait.
Take Chymotrypsin for example; the inactive form of it is called chymoprypsinogen which will be cleaved by trypsin to result in pi-Chymostrypin, which will in turn cleave others of its kind and result in the final alpha-chymotrypsin. This will control when and where chymotrypsin cleaves, so it does not cleave in the wrong environments or time.
Chymotrypsinogen is the zymogen (inactive precursor) to the digestive enzyme trypsin. It is synthesized in the pancreas. They are stored in zymogen granules in the acinar cells of the pancreas. When a nerve impulse reaches the pancreas, it stimulates the granules to release chymotrypsinogen into the lumen leading to the small intestine. Chymotrypsin activated when the peptide bond between arginine 15 and isoleucine 16 is cleaved by trypsin. This creates two pi-chymotrypsin peptides. The pi-chymotrypsin then cleaves other pi-chymotrypsin peptides. The final result is the active form alpha-chymotrypsin which is made up of three chains.
Another example of proteolytic activation of enzymes can be seen in blood clotting. When trauma occurs to tissue, it starts a blood-clotting cascade. The activation of one clotting factor triggers the activation of other triggers which creates an amplified effect that allows the body to quickly respond to the injury. When blood vessels rupture, it activates tissue factor (TF). TF then activates thrombin, a protease that cleaves fibrinogen, the zymogen of fibrin. When fibrin is formed, it polymerizes to form clots.
Controlling Enzyme Present
This form of regulation is controlled with transcription, different protein will bind to specific DNA sequences to regulate the transcription of certain segments of DNA. The transcription of a certain enzymatic genes can be adjusted to the changes in a cells environment. This will affect the amount of enzyme present in the system, an thus regulate the catalysis of the reaction.