Structural Biochemistry/Enzyme Regulation/Feedback inhibition

Feedback inhibition is the phenomenon where the output of a process is used as an input to control the behavior of the process itself, oftentimes limiting the production of more product. Although negative feedback is used in the context of inhibition, negative feedback may also be used for promoting a certain process. An everyday example of negative feedback is the cruise control in automobiles. The faster a car goes above the cruise control speed, the stronger the brakes are applied to slow the car down. If the car is going too slowly, more gas is fed to the engine to speed the car up. In a biological context, the more product produced by the enzyme, the more inhibited the enzyme is towards creating additional product.

Many enzyme catalyzed reactions are carried out through a biochemical pathway. In these pathways, the product of one reaction becomes the substrate for the next reaction. At the end of the pathway, a desired product is synthesized. In order to tightly regulate the concentration of that product, the biochemical pathway needs to be shut down. This is done through feedback inhibition. The product of the final reaction in that pathway reacts with an enzyme somewhere along the pathway at the enzyme's allosteric site, changing the conformation of the enzyme. That enzyme can no longer bind to its substrate as effectively due to the conformational change, closing down that pathway and stopping the final product from synthesizing. The higher the concentration of the final product, the more likely that product will bind to the allosteric site of the enzyme, shutting down that pathway.

There are many intermediates and pathways in feedback inhibition. Often the final product Z will inhibit the initial reactant A.

Mechanism of Negative Feedback

Each metabolic reaction or process is regulated by several enzymes. These enzymes control the rate of these reactions and thus are fundamental in maintaining homeostasis. Below is a universal map of how this type of inhibition works. We will start with a substrate that is attacked by enzyme 1, forming product A which then acts as the substrate for enzyme 2 forming product B. Product B then becomes the substrate for the attack of enzyme 3 forming our final product.

substrate ---enzyme 1--> product A ----enzyme 2---> product B ----enzyme 3----> Final Product

Keep in mind that the final product is usually something the body uses up and is necessary for homeostasis. In this reaction, the purpose of the intermediates, product A and product B, is to move the reaction along to reach the final product therefore the inhibition mechanism does not start at these intermediates but at the final product. As the amount of final product becomes elevated, the system imposes a halting effect on enzyme 1, slowing down the production of intermediates A and B, reducing the formation of the final product. When levels of the final product fall below a threshold, the effect of negative feedback diminishes and enzyme 1 is reactivated and the reaction process will be started again.

So what forces are responsible for creating these feedback responses? There are several regulators that affect a given process. Hormones and chemical signals produced and distributed by the hypothalamus and pituitary glands, for example, are the regulators that act in feedback loops. To illustrate the concept of this section, let's investigate the regulation of blood sugar levels. The hormones insulin and glucagon are two regulators that are intimately related in regulating sugar levels. Insulin is responsible for triggering different cells in the body to absorb glucose from the blood and to store the excess as glycogen for later utility. Conversely, glucagon's function is to convert glycogen supply into glucose. When blood glucose level is too low, the alpha cells of the islets of Langerhans in the pancreas release glucagon. Glucagon subsequently activates the conversion of glycogen to glucose until the sugar level in the blood is back to its normal state. When blood glucose is too high the beta cells of the islets of Langerhans release insulin which causes cells in the body to take up sugar quickly, lowering the blood sugar level to its normal level.

Positive Feedback

In contrast to negative feedback, positive feedback occurs when an output is used as a signal to increase further response of the output. In other words, if process A results in consequence B, B reinforces process A, resulting in a cascade where more of B occurs, which causes more of A to occur, and so on. An example of a positive feedback loop is evolution, where an organism evolves and becomes better at hunting prey, for example, prey evolve better defense mechanism like faster running, which causes predators to adapt by evolving better chasing skills, and so forth. Note that a positive or negative feedback mechanism is not necessarily beneficial or harmful; they only refer to the mechanism by which inhibition or propagation occurs.

Examples of Feedback Inhibition

Feedback inhibition controls the production of amino acids. The benefits of feedback inhibition are that the building blocks such as 3-phosphoglycerate, which is crucial to other processes such as the Calvin cycle and glycolysis, are used optimally and without waste.

Note: the structure of 3-phosphoglycerate is shown here

Feedback inhibition also controls nucleotide production. The pyrimidines (Thymine, Cytosine, and Uracil) have different pathways and feedback mechanisms than the Purines (Adenine and Guanine). Aspartate transcarbamoylase [1] regulates pyrimidine synthesis in bacteria. The regulation for purine production begins as PRPP or 5-phosphoribosyl-1-pyrophosphate which is converted into Phosphoribosylamine. This pathway is inhibited by IMP, AMP, and GMP. Then Phosphribosylamine is converted into IMP. IMP is a common precursor to both Adenosine and Guanine. The pathways from IMP to the Adenosine and Guanine precursors of AMP and GMP, respectively, are separated. IMP to AMP is inhibited by AMP(adenosine precursor) and IMP to GMP(guanine precursor) are inhibited by GMP, thus the products are inhibiting the precursors.

Cholesterol production in the liver is catalyzed when cholesterol levels are low. This is done on the mRNA level of transcription through a transcription factor called the sterol regulatory element binding protein or SREBP. The role of SREBP is to increase the rate of transcription of mRNA by binding to a short DNA strand called sterol regulatory element or SRE. Conversely translation of reductase is inhibited by consumed cholesterol and other derivatives.

Negative feedback results in inhibition, but another powerful tool in biological systems is the positive feedback cycle. This process is the opposite of negative feedback. We can find an example of it in catalytic cascade processes, such as blood clotting. An initial factor will begin the cascade, say catalyzing or activating proteases, and which each step, additional steps will follow due to the chain reaction. Each step will amplify the signal first given, until it reaches its destination or purpose. In this sense, a very little amount of the initial factor is needed since the steps following provides efficient magnification.

Feedback inhibition is a form of allosteric regulation in which the final product of a sequence of enzymatic reactions accumulates in abundance. With too much of this product produced, the final product binds to an allosteric site on the first enzyme in the series of reactions to inhibit its activity. This halts the reaction at the first step so that no more excess product is produced. In the images above, the second to last product is the one that halts the reaction by biding allosterically to the active site on the first enzyme. This is done to illustrate that not all feedback inhibition is exactly clear cut. Different processes will be regulated differently depending on a variety of factors such as the enzymes and substrates involved, and the conditions in which the reaction takes place.

A series of enzymatic reactions involving multiple enzymes and substrates to reach a final product

A series of enzymatic reactions halted by feedback inhibition when the second product in the sequence binds allosterically to the first enzyme to inhibit its catalytic activity

ATCase

Aspartate transcarbanoylase catalyzes the first step in the synthesis of pyrimidines. As mentioned above, after aspartate transcarbamoylase catalyzes the committed step, also known as,the condensation of aspartate and carbamoyl phosphate takes place to form N-carbamoylaspartate, in pyrimidine synthesis. This will yield pyrimitdine nucleotides such as cytidine triphosphate (CTP)(See Figure Below).

ATCase Reaction

CTP Inhibits ATCase

ATCase Sigmoidal Kinetics

Modifications of Cysteine Residues

The molecule CTP is also known to be used in feedback inhibition in conjunction with aspartate transcarbamoylase (ATCase)(See Figure: CTP Inhibits ATCase). CTP, which is the final product of the metabolic pathway started by ATCase, inhibits ATCase when there is CTP in excess. When there is excess CTP, the enzyme activity decreases which explains why CTP favors the T state which is less active. This type of inhibition regulates that N-carbamoylaspartate and other subsequent intermediates in the pathway are not unnecessarily formed when the concentration of pyrimidines is large.

Allosterically regulated enzymes, such as ATCase do not follow Michaelis-Menten Kinetics. Allosteric enzymes are differentiated from other enzymes due their response to changes in substrace concentration levels and their susceptibility to regulation by other molecules. The plot of rate of product formation as a function of substrate concentration for ATCase differs from that expected for enzymes that obey the Michaelis-Menten kinetics. Instead, the curve for ATCase is in the form of a sigmoidal curve, which is due to the fact that binding of substrate to one active site of the enzyme increases the activity at the other active sites. This means that they enzyme has cooperative properties, similar to that of hemoglobin, the protein in our blood that transports oxygen molecules throughout our body. (See Figure: ATCase Sigmoidal Kinetics).

CTP has a structure that is different from the substrates of the reaction. Thus, CTP must bind the different active sites called regulatory sites.

p-Hydroxymercuribenzoate separates the catalytic (c chain) and the regulatory subunits (r chain) of ATCase, in which the p-hydroxybenzoate reacts with sulfhydryl groups on the cysteine residues in ATCase.(See Figure: modification of cysteine residues). Ultracentrifugation studies have shown that mercurials can dissociate ATCase into these two kinds of subunits, in which the subunits can be separated by ion-exchange chromatography. Ion-exchange chromatography is effective in this case because the subunits differ in their charge. The subunits can also be separated by centrifugation in a sucrose density gradient since the subunits differ in size. ATCase is 6subunits of two trimers . Here is a regulatory dimer and a catalytic trimer. CTP is an allosteric inhibitor, and it binds to regulatory subunits of the less active T state, which is favored by CTP binding. CTP decreases the activity of the enzyme. ATP competes with CTP because ATP stimulates the reaction by binding to where CTP will bind.

Two c chains are stacked on one another and linked to three r chains. The contact between the r chains and c chains are stabilized by a Zinc ion bound to four cysteine residues. To separate the r and c chains, the mercurial compound p-Hydroxmercuribenoate can be used. This compound can separate the chains because it has mercury, which strongly binds to cysteine residues, displays the Zinc ion, and destabilizes it.

For more information on ATCase: [2]

References

Berg, Jeremy M., Lubert Stryer, and John L. Tymoczko. "The BioSynthesis of Amino Acids." Biochemistry. 6th ed. 697-98. 723-724. 742-743.

http://www.scribd.com/doc/8639011/161-Negative-Feedback-Mechanisms.