Design of Allosterically Regulated Protein Catalysts

Design of Allosterically Regulated Protein Catalysts

Enzymes are selective and efficient in catalyzing chemical reactions. Enzymatic actions can be controlled using several stimuli such as light, changing of pH levels, protein or ligand binding and controlling the electrostatic potential. Enzymes need to be regulated to have an effect on their ability to catalyze and this is done through their allosteric domain where you can perform conformational changes. This makes it possible to have allosterically regulated proteins that have many conformations which possess different properties. Protein engineering and design has advanced and this has allowed the formation of catalysts whose function can be controlled. However there is a challenge of translating the signal from an input domain to the output domain in an allosterically regulated enzyme. This article discusses on ways of countering this challenge using various techniques.

Modification of the Linker Domain

Allosterically regulated catalysts can be created through have two protein domains that are connected.  Protein domains can be linked using various techniques such as domain insertion, control of enzyme assembly, and domain swapping. Domain insertion involves the fuse of two proteins where their properties combine. This allows for formation of fusion proteins in which a protein’s catalytic function can be connected to a binding event of a different protein. Domain insertion is not an easy design making its success to create allosterically regulated proteins limited. This is because the identification of sites that can tolerate domain insertion is difficult. To ensure this technique is successful, one protein is randomly inserted into different linkers then allosteric regulation is tested to measure the most efficient. Various domain insertion studies have been conducted such as linking a maltose-binding protein with β-lactamase (BLA) and regulation of focal adhesion kinase (FAK) using rapamycin.

Domain swapping involves the exchange of domains between homologous proteins. Some proteins have different functions but they contain similar signaling mechanisms and structures and this makes PAS domains to be interchangeable. The PAS signaling proteins family comprises of many members whose sensor and effector domain combination are different. This makes it possible to use them in designing allosterically designed enzymes. The control of enzyme assembly is a technique that deals with splitting enzymes then reassembling them. An enzyme is split into two parts which are the fused with an allosteric domain. This leads to the creation of a catalyst where a ligand controls it in terms of its concentration levels. When modifying using the linker domain it is important to ensure that the domains are close to one another for efficient coupling. It is important to understand the linker regions regarding their location and structure as this is necessary for the design to be created.

Creation of a New Catalytic Site

The approach involves designing a novel catalytic activity that has a link with a ligand-promoted conformational change and this involves the use of a de novo design. This design of allosterically regulated catalysts has been successful. Experiments in this section involves calcium dependent catalysts used in retroaldol reaction, ester hydrolysis and Kemp elimination. Kemp elimination involves a reaction that is catalyzed by separating a proton from benzisoxazole and splitting of isoxazole ring where yellow 2-cyanophenolate is formed. This reaction requires that the basicity of the carboxylate to be high and hence carboxylic acids are dissolved in organic solvents and they are used as catalysts for the reaction. The kemp eliminase activity occurs when a carboxylate residue is introduced in a protein hydrophobic pocket.

Ester hydrolysis reactions are used in many places such as in biofuel production and chemical synthesis. An example of this reaction is the creation of a yellow substance from ρ-nitrophenyl esters and it is used to determine whether an esterase activity could be performed by allosterically regulated CaM. A retroaldol reaction can be exemplified where a single reactive lysine is strategically placed into calmodulin’s hydrophobic pocket. This reaction results in the formation of an allosterically regulated catalyst known as AlleyCatR which can be used in creation of biocompatible metal sensors. The success of this approach that involves a new formation of a catalytic site is dependent on the ability of creating catalytic functionality that is efficient

Modification of Existing or Creation of New Allogesteric Sites

When a sensor is undergoing transition from a situation where ligands were not present to a ligand bound state, the sensor requires to undergo conformational changes. This transition involves the addition of a ligand binding into an enzyme or having an allosterically controlled enzyme binding with a ligand that it chooses. Some of the examples used in this section include chemical rescue, redesign of an existing allosteric site, modular creation of an allosteric site and photocaged systems. The chemical rescue approach involves the partial replacement of the functions of amino acids by a small molecule. A residue involved in catalysis is mutated and then a molecule is added which will complement the functionality of the mutated residue. Another way to perform the chemical rescue involves the active site destabilization of the enzyme. This leads to the creation of a cavity that is filled by a small molecule and this restores the functionality of the enzyme.

The redesigning of an existing allosteric site can be explained by showing how β-lactamase is modified so that it can respond to sucrose. Another experiment to show the same can be the reprogramming of CuSeCat protein so that it can bind with lanthanide ions. Modular creation of an allosteric site involves the allosteric control of enzymes using metabolites. Photocaged systems, on the other hand, involve caging that helps in regulating enzymatic activities. This approach uses a nonreversible approach that can only be turned once. This makes the presence of photocaged enzymes and experiments to be scarce. However developments have been made where the reaction in this approach can be turned by light that is the light is used in uncaging a residue and its functionality is restored.

In conclusion, designing allosterically regulated protein is no easy task and the three approaches in doing are linker, allosteric or catalytic sites. Linking is helpful in achieving the regulation of catalysis using an external stimulus because of the presence of efficient sensors and catalysts. A catalytic site is also utilized in allosteric regulation and also creation of different abilities for the introduced functions. Allosteric sites are complex to design and this has seen the only successful example being chemical rescue. More studies should be directed to protein design and engineering to have more designs of allosterically regulated enzymes.

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