Active site

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Active_site 

 

Organisation of enzyme structure and lysozyme example. Binding sites in blue, catalytic site in red and peptidoglycan substrate in black. (PDB: 9LYZ​)

In biology, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) and residues that catalyse a reaction of that substrate (catalytic site). Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

Each active site is evolved to be optimised to bind a particular substrate and catalyse a particular reaction, resulting in high specificity. This specificity is determined by the arrangement of amino acids within the active site and the structure of the substrates. Sometimes enzymes also need to bind with some cofactors to fulfil their function. The active site is usually a groove or pocket of the enzyme which can be located in a deep tunnel within the enzyme, or between the interfaces of multimeric enzymes. An active site can catalyse a reaction repeatedly as residues are not altered at the end of the reaction (they may change during the reaction, but are regenerated by the end). This process is achieved by lowering the activation energy of the reaction, so more substrates have enough energy to undergo reaction.

Binding site

Diagram of the lock and key hypothesis

 

Diagram of the induced fit hypothesis

Main article: Binding site

Usually, an enzyme molecule has only two active sites, and the active sites fit with one specific type of substrate. An active site contains a binding site that binds the substrate and orients it for catalysis. The orientation of the substrate and the close proximity between it and the active site is so important that in some cases the enzyme can still function properly even though all other parts are mutated and lose function.

Initially, the interaction between the active site and the substrate is non-covalent and transient. There are four important types of interaction that hold the substrate in a defined orientation and form an enzyme-substrate complex (ES complex): hydrogen bonds, van der Waals interactions, hydrophobic interactions and electrostatic force interactions. The charge distribution on the substrate and active site must be complementary, which means all positive and negative charges must be cancelled out. Otherwise, there will be a repulsive force pushing them apart. The active site usually contains non-polar amino acids, although sometimes polar amino acids may also occur. The binding of substrate to the binding site requires at least three contact points in order to achieve stereo-, regio-, and enantioselectivity. For example, alcohol dehydrogenase which catalyses the transfer of a hydride ion from ethanol to NAD⁺ interacts with the substrate methyl group, hydroxyl group and the pro-(R) hydrogen that will be abstracted during the reaction.

In order to exert their function, enzymes need to assume their correct protein fold (native fold) and tertiary structure. To maintain this defined three-dimensional structure, proteins rely on various types of interactions between their amino acid residues. If these interactions are interfered with, for example by extreme pH values, high temperature or high ion concentrations, this will cause the enzyme to denature and lose its catalytic activity.

A tighter fit between an active site and the substrate molecule is believed to increase the efficiency of a reaction. If the tightness between the active site of DNA polymerase and its substrate is increased, the fidelity, which means the correct rate of DNA replication will also increase. Most enzymes have deeply buried active sites, which can be accessed by a substrate via access channels.

There are three proposed models of how enzymes fit their specific substrate: the lock and key model, the induced fit model, and the conformational selection model. The latter two are not mutually exclusive: conformational selection can be followed by a change in the enzyme's shape. Additionally, a protein may not wholly follow either model. Amino acids at the binding site of ubiquitin generally follow the induced fit model, whereas the rest of the protein generally adheres to conformational selection. Factors such as temperature likely influences the pathway taken during binding, with higher temperatures predicted to increase the importance of conformational selection and decrease that of induced fit.

Lock and key hypothesis

This concept was suggested by the 19th-century chemist Emil Fischer. He proposed that the active site and substrate are two stable structures that fit perfectly without any further modification, just like a key fits into a lock. If one substrate perfectly binds to its active site, the interactions between them will be strongest, resulting in high catalytic efficiency.

As time went by, limitations of this model started to appear. For example, the competitive enzyme inhibitor methylglucoside can bind tightly to the active site of 4-alpha-glucanotransferase and perfectly fits into it. However, 4-alpha-glucanotransferase is not active on methylglucoside and no glycosyl transfer occurs. The Lock and Key hypothesis cannot explain this, as it would predict a high efficiency of methylglucoside glycosyl transfer due to its tight binding. Apart from competitive inhibition, this theory cannot explain the mechanism of action of non-competitive inhibitors either, as they do not bind to the active site but nevertheless influence catalytic activity.

Induced fit hypothesis

Daniel Koshland's theory of enzyme-substrate binding is that the active site and the binding portion of the substrate are not exactly complementary. The induced fit model is a development of the lock-and-key model and assumes that an active site is flexible and changes shape until the substrate is completely bound. This model is similar to a person wearing a glove: the glove changes shape to fit the hand. The enzyme initially has a conformation that attracts its substrate. Enzyme surface is flexible and only the correct catalyst can induce interaction leading to catalysis. Conformational changes may then occur as the substrate is bound. After the reaction products will move away from the enzyme and the active site returns to its initial shape. This hypothesis is supported by the observation that the entire protein domain could move several nanometers during catalysis. This movement of protein surface can create microenvironments that favour the catalysis.

Conformational selection hypothesis

This model suggests that enzymes exist in a variety of conformations, only some of which are capable of binding to a substrate. When a substrate is bound to the protein, the equilibrium in the conformational ensemble shifts towards those able to bind ligands (as enzymes with bound substrates are removed from the equilibrium between the free conformations).

Types of non-covalent interactions

Positively charged sodium ion and negatively charged fluoride ion attract each other to form sodium fluoride under electrostatic interaction.

 

Hydrogen bond between two water molecules.

 

Van der Waals force between two acetone molecules. The lower acetone molecule contains a partially negative oxygen atom that attracts partially positive carbon atom in the upper acetone.

 

Hydrophobic and hydrophilic groups tend to assemble with the same kind of molecules.

Electrostatic interaction: In an aqueous environment, the oppositely charged groups in amino acid side chains within the active site and substrates attract each other, which is termed electrostatic interaction. For example, when a carboxylic acid (R-COOH) dissociates into RCOO⁻ and H⁺ ions, COO⁻ will attract positively charged groups such as protonated guanidine side chain of arginine.

Hydrogen bond: A hydrogen bond is a specific type of dipole-dipole interaction between a partially positive hydrogen atom and a partially negative electron donor that contain a pair of electrons such as oxygen, fluorine and nitrogen. The strength of hydrogen bond depends on the chemical nature and geometric arrangement of each group.

Van der Waals force: Van der Waals force is formed between oppositely charged groups due to transient uneven electron distribution in each group. If all electrons are concentrated at one pole of the group this end will be negative, while the other end will be positive. Although the individual force is weak, as the total number of interactions between the active site and substrate is massive the sum of them will be significant.

Hydrophobic interaction: Non-polar hydrophobic groups tend to aggregate together in the aqueous environment and try to leave from polar solvent. These hydrophobic groups usually have long carbon chain and do not react with water molecules. When dissolving in water a protein molecule will curl up into a ball-like shape, leaving hydrophilic groups in outside while hydrophobic groups are deeply buried within the centre.

Catalytic site

Main article: Enzyme catalysis

See also: Catalytic triad

The enzyme TEV protease contains a catalytic triad of residues (red) in its catalytic site. The substrate (black) is bound by the binding site to orient it next to the triad. PDB: 1lvm​

Once the substrate is bound and oriented to the active site, catalysis can begin. The residues of the catalytic site are typically very close to the binding site, and some residues can have dual-roles in both binding and catalysis.

Catalytic residues of the site interact with the substrate to lower the activation energy of a reaction and thereby make it proceed faster. They do this by a number of different mechanisms including the approximation of the reactants, nucleophilic/electrophilic catalysis and acid/base catalysis. These mechanisms will be explained below.

Mechanisms involved in Catalytic process

Approximation of the reactant

During enzyme catalytic reaction, the substrate and active site are brought together in a close proximity. This approach has various purposes. Firstly, when substrates bind within the active site the effective concentration of it significantly increases than in solution. This means the number of substrate molecules involved in the reaction is also increased. This process also reduces the desolvation energy required for the reaction to occur. In solution substrate molecules are surrounded by solvent molecules and energy is required for enzyme molecules to replace them and contact with the substrate. Since bulk molecules can be excluded from the active site this energy output can be minimised. Next, the active site is designed to reorient the substrate to reduce the activation energy for the reaction to occur. The alignment of the substrate, after binding, is locked in a high energy state and can proceed to the next step. In addition, this binding is favoured by entropy as the energy cost associated with solution reaction is largely eliminated since solvent cannot enter active site. In the end, the active site may manipulate the Molecular orbital of the substrate into a suitable orientation to reduce activation energy.

The electrostatic states of substrate and active site must be complementary to each other. A polarized negatively charged amino acid side chain will repel uncharged substrate. But if the transition state involves the formation of an ion centre then the side chain will now produce a favourable interaction.

Covalent catalysis

Many enzymes including serine protease, cysteine protease, protein kinase and phosphatase evolved to form transient covalent bonds between them and their substrates to lower the activation energy and allow the reaction to occur. This process can be divided into 2 steps: formation and breakdown. The former step is rate-limit step while the later step is needed to regenerate intact enzyme.

Nucleophilic catalysis: This process involves the donation of electrons from the enzyme's nucleophile to a substrate to form a covalent bond between them during the transition state. The strength of this interaction depends on two aspects.: the ability of the nucleophilic group to donate electrons and the electrophile to accept them. The former one is mainly affected by the basicity(the ability to donate electron pairs) of the species while the later one is in regard to its pK_a. Both groups are also affected by their chemical properties such as polarizability, electronegativity and ionization potential. Amino acids that can form nucleophile including serine, cysteine, aspartate and glutamine.

Electrophilic catalysis: The mechanism behind this process is exactly same as nucleophilic catalysis except that now amino acids in active site act as electrophile while substrates are nucleophiles. This reaction usually requires cofactors as the amino acid side chains are not strong enough in attracting electrons.

Metal ions

Metal ions have multiple roles during the reaction. Firstly it can bind to negatively charged substrate groups so they will not repel electron pairs from active site's nucleophilic groups. It can attract negatively charged electrons to increase electrophilicity. It can also bridge between active site and substrate. At last, they may change the conformational structure of the substrate to favour reaction.

Acid/base catalysis

In some reactions, protons and hydroxide may directly act as acid and base in term of specific acid and specific base catalysis. But more often groups in substrate and active site act as Brønsted–Lowry acid and base. This is called general acid and general base theory. The easiest way to distinguish between them is to check whether the reaction rate is determined by the concentrations of the general acid and base. If the answer is yes then the reaction is the general type. Since most enzymes have an optimum pH of 6 to 7, the amino acids in the side chain usually have a pK_a of 4~10. Candidate include aspartate, glutamate, histidine, cysteine. These acids and bases can stabilise the nucleophile or electrophile formed during the catalysis by providing positive and negative charges.

Conformational distortion

Quantitative studies of enzymatic reactions often found that the acceleration of chemical reaction speed cannot be fully explained by existing theories like the approximation, acid/base catalysis and electrophile/nucleophile catalysis. And there is an obvious paradox: in reversible enzymatic reaction if the active site perfectly fits the substrates then the backward reaction will be slowed since products cannot fit perfectly into the active site. So conformational distortion was introduced and argues that both active site and substrate can undergo conformational changes to fit with each other all the time.

Preorganised active site complementarity to the transition state

This theory is a little similar to the Lock and Key Theory, but at this time the active site is preprogrammed to bind perfectly to substrate in transition state rather than in ground state. The formation of transition state within the solution requires a large amount of energy to relocate solvent molecules and the reaction is slowed. So the active site can substitute solvent molecules and surround the substrates to minimize the counterproductive effect imposed by the solution. The presence of charged groups with the active site will attract substrates and ensure electrostatic complementarity.

Examples of enzyme catalysis mechanisms

In reality, most enzyme mechanisms involve a combination of several different types of catalysis.

Glutathione reductase

the mechanism of glutathione reductase

The role of glutathione(GSH) is to remove accumulated reactive oxygen species which may damage cells. During this process, its thiol side chain is oxidised and two glutathione molecules are connected by a disulphide bond to form a dimer(GSSG). In order to regenerate glutathione the disulphide bond has to be broken, In human cells, this is done by glutathione reductase(GR).

Glutathione reductase is a dimer that contains two identical subunits. It requires one NADP and one FAD as the cofactors. The active site is located in the linkage between two subunits. The NADPH is involved in the generation of FADH-. In the active site, there are two cysteine residues besides the FAD cofactor and are used to break the disulphide bond during the catalytic reaction. NADPH is bound by three positively charged residues: Arg-218, His-219 and Arg-224.

The catalytic process starts when the FAD is reduced by NADPH to accept one electron and from FADH⁻. It then attacks the disulphide bond formed between 2 cysteine residues, forming one SH bond and a single S⁻ group. This S⁻ group will act as a nucleophile to attack the disulphide bond in the oxidised glutathione(GSSG), breaking it and forming a cysteine-SG complex. The first SG⁻ anion is released and then receives one proton from adjacent SH group and from the first glutathione monomer. Next the adjacent S⁻ group attack disulphide bond in cysteine-SG complex and release the second SG⁻ anion. It receives one proton in solution and forms the second glutathione monomer.

Chymotrypsin

Mechanism of peptide bond cleavage by chymotrypsin.

Chymotrypsin is a serine endopeptidase that is present in pancreatic juice and helps the hydrolysis of proteins and peptide. It catalyzes the hydrolysis of peptide bonds in L-isomers of tyrosine, phenylalanine, and tryptophan. In the active site of this enzyme, three amino acid residues work together to form a catalytic triad which makes up the catalytic site. In chymotrypsin, these residues are Ser-195, His-57 and Asp-102.

The mechanism of chymotrypsin can be divided into two phases. First, Ser-195 nucleophilically attacks the peptide bond carbon in the substrate to form a tetrahedral intermediate. The nucleophilicity of Ser-195 is enhanced by His-57, which abstracts a proton from Ser-195 and is in turn stabilised by the negatively charged carboxylate group (RCOO⁻) in Asp-102. Furthermore, the tetrahedral oxyanion intermediate generated in this step is stabilised by hydrogen bonds from Ser-195 and Gly-193.

In the second stage, the R'NH group is protonated by His-57 to form R'NH₂ and leaves the intermediate, leaving behind the acylated Ser-195. His-57 then acts as a base again to abstract one proton from a water molecule. The resulting hydroxide anion nucleophilically attacks the acyl-enzyme complex to form a second tetrahedral oxyanion intermediate, which is once again stabilised by H bonds. In the end, Ser-195 leaves the tetrahedral intermediate, breaking the CO bond that connected the enzyme to the peptide substrate. A proton is transferred to Ser-195 through His-57, so that all three amino acid return to their initial state.

Unbinding

Substrate unbinding is influenced by various factors. Larger ligands generally stay in the active site longer, as do those with more rotatable bonds (although this may be a side effect of size). When the solvent is excluded from the active site, less flexible proteins result in longer residence times. More hydrogen bonds shielded from the solvent also decrease unbinding.

Cofactors

Redox states of Flavin.

Main article: Cofactor (biochemistry)

Enzymes can use cofactors as ‘helper molecules’. Coenzymes are referred to those non-protein molecules that bind with enzymes to help them fulfill their jobs. Mostly they are connected to the active site by non-covalent bonds such as hydrogen bond or hydrophobic interaction. But sometimes a covalent bond can also form between them. For example, the heme in cytochrome C is bound to the protein through thioester bond. In some occasions, coenzymes can leave enzymes after the reaction is finished. Otherwise, they permanently bind to the enzyme. Coenzyme is a broad concept which includes metal ions, various vitamins and ATP. If an enzyme needs coenzyme to work itself, it is called an apoenzyme. In fact, it alone cannot catalyze reactions properly. Only when its cofactor comes in and binds to the active site to form holoenzyme does it work properly.

One example of the coenzyme is Flavin. It contains a distinct conjugated isoalloxazine ring system. Flavin has multiple redox states and can be used in processes that involve the transfer of one or two electrons. It can act as an electron acceptor in reaction, like the oxidation of NAD to NADH, to accept two electrons and form 1,5-dihydroflavin. On the other hand, it can form semiquinone(free radical) by accepting one electron, and then converts to fully reduced form by the addition of an extra electron. This property allows it to be used in one electron oxidation process.

Inhibitors

Main article: Enzyme inhibitor

Inhibitors disrupt the interaction between enzyme and substrate, slowing down the rate of a reaction. There are different types of inhibitor, including both reversible and irreversible forms.

Competitive inhibitors are inhibitors that only target free enzyme molecules. They compete with substrates for free enzyme acceptor and can be overcome by increasing the substrate concentration. They have two mechanisms. Competitive inhibitors usually have structural similarities to the substrates and or ES complex. As a result, they can fit into the active site and trigger favourable interactions to fill in the space and block substrates from entry. They can also induce transient conformational changes in the active site so substrates cannot fit perfectly with it. After a short period of time, competitive inhibitors will drop off and leave the enzyme intact.

Inhibitors are classified as non-competitive inhibitors when they bind both free enzyme and ES complex. Since they do not compete with substrates for the active site, they cannot be overcome by simply increasing the substrate concentration. They usually bind to a different site on the enzyme and alter the 3-dimensional structure of the active site to block substrates from entry or leaving the enzyme.

Irreversible inhibitors are similar to competitive inhibitors as they both bind to the active site. However, irreversible inhibitors form irreversible covalent bonds with the amino acid residues in the active site and never leave. Therefore, the active site is occupied and the substrate cannot enter. Occasionally the inhibitor will leave but the catalytic site is permanently altered in shape. These inhibitors usually contain electrophilic groups like halogen substitutes and epoxides. As time goes by more and more enzymes are bound by irreversible inhibitors and cannot function anymore.

	Example	Binds active site?	Reduces rate of reaction?
Competitive reversible inhibitor	HIV protease inhibitors	Yes	Yes
Non-competitive reversible inhibitor	Heavy metals such as lead and mercury	No	Yes
Irreversible inhibitor	Cyanide	Yes	Yes

Examples of competitive and irreversible enzyme inhibitors

Competitive inhibitor: HIV protease inhibitor

Indinavir, an HIV protease inhibitor.jpg

HIV protease inhibitors are used to treat patients having AIDS virus by preventing its DNA replication. HIV protease is used by the virus to cleave Gag-Pol polyprotein into 3 smaller proteins that are responsible for virion assembly, package and maturation. This enzyme targets the specific phenylalanine-proline cleave site within the target protein. If HIV protease is switched off the virion particle will lose function and cannot infect patients. Since it is essential in viral replication and is absent in healthy human, it is an ideal target for drug development.

HIV protease belongs to aspartic protease family and has a similar mechanism. Firstly the aspartate residue activates a water molecule and turns it into a nucleophile. Then it attacks the carbonyl group within the peptide bond (NH-CO) to form a tetrahedral intermediate. The nitrogen atom within the intermediate receives a proton, forming an amide group and subsequent rearrangement leads to the break down of the bond between it and the intermediate and forms two products.

Inhibitors usually contain a nonhydrolyzable hydroxyethylene or hydroxyethylamine groups that mimic the tetrahedral intermediate. Since they share a similar structure and electrostatic arrangement to the transition state of substrates they can still fit into the active site but cannot be broken down, so hydrolysis cannot occur.

Non-competitive inhibitor: Strychnine

Strychnine is a neurotoxin that causes death by affecting nerves that control muscular contraction and cause respiration difficulty. The impulse is transmitted between the synapse through a neurotransmitter called acetylcholine. It is released into the synapse between nerve cells and binds to receptors in the postsynaptic cell. Then an action potential is generated and transmitted through the postsynaptic cell to start a new cycle.

Glycine can inhibit the activity of neurotransmitter receptors, thus a larger amount of acetylcholinesterase is required to trigger an action potential. This makes sure that the generation of nerve impulses is tightly controlled. However, this control is broken down when strychnine is added. It inhibits glycine receptors(a chloride channel) and a much lower level of neurotransmitter concentration can trigger an action potential. Nerves now constantly transmit signals and cause excessive muscular contraction, leading to asphyxiation and death.

Irreversible inhibitor:Diisopropyl fluorophosphate

Irreversible inhibition of a serine protease by DIPF.

Diisopropyl fluorophosphate (DIFP) is an irreversible inhibitor that blocks the action of serine protease. When it binds to the enzyme a nucleophilic substitution reaction occurs and releases one hydrogen fluoride molecule. The OH group in the active site acts as a nucleophile to attack the phosphorus in DIFP and form a tetrahedral intermediate and release a proton. Then the P-F bond is broken, one electron is transferred to the F atom and it leaves the intermediate as F⁻ anion. It combines with a proton in solution to form one HF molecule. A covalent bond formed between the active site and DIFP, so the serine side chain is no longer available to the substrate.

In drug discovery

Identification of active sites is crucial in the process of drug discovery. The 3-D structure of the enzyme is analysed to identify active site residues and design drugs which can fit into them. Proteolytic enzymes are targets for some drugs, such as protease inhibitors, which include drugs against AIDS and hypertension. These protease inhibitors bind to an enzyme's active site and block interaction with natural substrates. An important factor in drug design is the strength of binding between the active site and an enzyme inhibitor. If the enzyme found in bacteria is significantly different from the human enzyme then an inhibitor can be designed against that particular bacterium without harming the human enzyme. If one kind of enzyme is only present in one kind of organism, its inhibitor can be used to specifically wipe them out.

Active sites can be mapped to aid the design of new drugs such as enzyme inhibitors. This involves the description of the size of an active site and the number and properties of sub-sites, such as details of the binding interaction. Modern database technology called CPASS (Comparison of Protein Active Site Structures) however allows the comparison of active sites in more detail and the finding of structural similarity using software.

Application of enzyme inhibitors

	Example	Mechanism of action
Anti-bacterial agent	Penicillin	The bacterial cell wall is composed of peptidoglycan. During bacterial growth the present crosslinking of peptidoglycan fibre is broken, so new cell wall monomer can be integrated into the cell wall. Penicillin works by inhibiting the transpeptidase which is essential for the formation of crosslinks, so the cell wall is weakened and will burst open due to turgor pressure.
Anti-fungi agent	Azole	Ergosterol is a sterol that forms the cell surface membrane of the fungi. Azole can inhibit its biosynthesis by inhibiting the Lanosterol 14 alpha-demethylase, so no new ergosterol is produced and harmful 14α-lanosterol is accumulated within the cell. Also, azole may generate reactive oxygen species.
Anti-viral agent	Saquinavir	HIV protease is needed to cleave Gag-Pol polyprotein into 3 individual proteins so they can function properly and start viral packaging process. HIV protease inhibitors like Saquinavir inhibit it so no new mature viral particle can be made.
Insecticides	Physostigmine	In the animal nervous system, Acetylcholinesterase is required to break down the neurotransmitter acetylcholine into acetate and choline. Physostigmine binds to its active site and inhibits it, so impulse signal cannot be transmitted through nerves. This results in the death of insects as they lose control of muscle and heart function.
Herbicides	Cyclohexanedione	Cyclohexanedione targets the Acetyl-CoA carboxylase which is involved in the first step of fat synthesis: ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. Lipids are important in making up the cell membrane.

Allosteric sites

Main article: Allosteric regulation

A – Active site B – Allosteric site C – Substrate D – Inhibitor E – Enzyme. This is a diagram of allosteric regulation of an enzyme. When inhibitor binds to the allosteric site the shape of active site is altered, so substrate cannot fit into it

An allosteric site is a site on an enzyme, unrelated to its active site, which can bind an effector molecule. This interaction is another mechanism of enzyme regulation. Allosteric modification usually happens in proteins with more than one subunit. Allosteric interactions are often present in metabolic pathways and are beneficial in that they allow one step of a reaction to regulate another step. They allow an enzyme to have a range of molecular interactions, other than the highly specific active site.

Artificial enzyme

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Artificial_enzyme#Nanozymes 

Schematic drawing of artificial phosphorylase

An artificial enzyme is a synthetic, organic molecule or ion that recreates some function of an enzyme. The area promises to deliver catalysis at rates and selectivity observed in many enzymes.

History

Enzyme catalysis of chemical reactions occur with high selectivity and rate. The substrate is activated in a small part of the enzyme's macromolecule called the active site. There, the binding of a substrate close to functional groups in the enzyme causes catalysis by so-called proximity effects. It is possible to create similar catalysts from small molecule by combining substrate-binding with catalytic functional groups. Classically artificial enzymes bind substrates using receptors such as cyclodextrin, crown ethers, and calixarene.

Artificial enzymes based on amino acids or peptides as characteristic molecular moieties have expanded the field of artificial enzymes or enzyme mimics. For instance, scaffolded histidine residues mimics certain metalloproteins and -enzymes such as hemocyanin, tyrosinase, and catechol oxidase).

Artificial enzymes have been designed from scratch via a computational strategy using Rosetta. In December 2014, it was announced that active enzymes had been produced that were made from artificial molecules which do not occur anywhere in nature. In 2016, a book chapter entitled "Artificial Enzymes: The Next Wave" was published.

Nanozymes

Nanozymes are nanomaterials with enzyme-like characteristics. They have been widely explored for various applications, such as biosensing, bioimaging, tumor diagnosis and therapy, antibiofouling.

1990s

In 1996 and 1997, Dugan et al. discovered the superoxide dismutase (SOD) mimicking activities of fullerene derivatives.

2000s

A "short review" article appeared in 2005. It attributed the term "nanozyme"s to "analogy with the activity of catalytic polymers (synzymes)", based on the "outstanding catalytic efficiency of some of the functional nanoparticles synthesized". The term was coined the previous year by Flavio Manea, Florence Bodar Houillon, Lucia Pasquato, and Paolo Scrimin. In 2006, nanoceria (i.e., CeO₂ nanoparticles) was reported as observed, in rat experiments, preventing retinal degeneration induced by intracellular peroxides (toxic reactive oxygen intermediates). This was seen as indicating a possible route to an eventual treatment for causes of blindness. In 2007 intrinsic peroxidase-like activity of ferromagnetic nanoparticles was reported by Yan Xiyun and coworkers as suggesting a wide range of applications in, for example, medicine and environmental chemistry, and the authors reported an immunoassay based on this property. Hui Wei and Erkang Wang then (2008) used this mimetic property of easily prepared magnetic nanoparticles (MNP) to demonstrate analytical applications to bioactive molecules, describing a colorimetric assay for hydrogen peroxide (H
₂O
₂) and a sensitive and selective platform for glucose detection.

2010s

As of 2016 review articles are appearing every year, in a range of journals. A book-length treatment appeared in 2015, described as providing "a broad portrait of nanozymes in the context of artificial enzyme research", and a 2016 Chinese book on "Enzyme Engineering" included a chapter on "Nanozymes".

Colorimetric applications of peroxidase mimesis in different preparations were reported in 2010 and 2011, detecting, respectively, glucose (via carboxyl‐modified graphene oxide) and single-nucleotide polymorphisms (via hemin−graphene hybrid nanosheets, and without labelling), with advantages in both cases of cost and convenience. A use of colour to visualise tumour tissues was reported in 2012, using the peroxidase mimesis of MNP coated with a protein which recognises cancer cells and binds to them.

Also in 2012, nanowires of vanadium pentoxide (vanadia, V₂O₅) were shown to suppress marine biofouling by mimicry of vanadium haloperoxidase, with anticipated ecological benefits. A study at a different centre two years later reported V₂O₅ showing mimicry of glutathione peroxidase, in in-vitro mammalian cells, suggesting future therapeutic application. The same year, 2014, it was reported that a carboxylated fullerene (C3) was neuroprotective post-injury in an in-vivo primate model of Parkinson's disease.

In 2015, a supramolecular nanodevice was proposed for bioorthogonal regulation of a transitional-metal nanozyme, based on encapsulating the nanozyme in a monolayer of hydrophilic gold nanoparticles, alternatively isolating it from the cytoplasm or allowing access, according to a gatekeeping receptor molecule controlled by competing guest species; the device is of biomimetic size and was reported as successful within the living cell, controlling pro-fluorophore and prodrug activation processes: it was suggested for imaging and therapeutic applications. A facile process for producing Cu(OH)
₂ supercages was reported, and a demonstration of their intrinsic peroxidase-mimicry. A scaffolded "INAzyme" ("integrated nanozyme") arrangement was described, locating hemin (a peroxidase mimic) with glucose oxidase (GOx) in sub-micron proximity, providing a fast and efficient enzyme cascade reported as monitoring cerebral brain-cell glucose dynamically in vivo. A method of ionising hydrophobe-stabilised colloid nanoparticles was described, with confirmation of their enzyme mimicry in aqueous dispersion.

Field trials were announced of an MNP-amplified rapid low-cost strip test for Ebola virus, in West Africa. H
₂O
₂ was reported as displacing label DNA, adsorbed to nanoceria, into solution, where it fluoresces, providing a highly sensitive glucose test. Oxidase-like nanoceria has been used for developing self-regulated bioassays. Multi-enzyme mimicking Prussian blue was developed for therapeutics. A review on MOF based enzyme mimics was published. Histidine was used to modulate iron oxide nanoparticles' peroxidase mimicking activities. Gold nanoparticles' peroxidase mimicking activities were modulated via a supramolecular strategy for cascade reactions. A molecular imprinting strategy was developed to improve the selectivity of Fe3O4 nanozymes with peroxidase-like activity. A new strategy was developed to enhance the peroxidase mimicking activity of gold nanoparticles by using hot electrons. Researchers have designed gold nanoparticles (AuNPs) based integrative nanozymes with both SERS and peroxidase mimicking activities for measuring glucose and lactate in living tissues. Cytochrome c oxidase mimicking activity of Cu2O nanoparticles was modulated by receiving electrons from cytochrome c. Fe3O4 NPs were combined with glucose oxidase for tumor therapeutics. Manganese dioxide nanozymes have been used as cytoprotective shells. Mn3O4 Nanozyme for Parkinson's Disease (cellular model) was reported. Heparin elimination in live rats has been monitored with 2D MOF based peroxidase mimics and AG73 peptide. Glucose oxidase and iron oxide nanozymes were encapsulated within multi-compartmental hydrogels for incompatible tandem reactions. A cascade nanozyme biosensor was developed for detection of viable Enterobacter sakazakii. An integrated nanozyme of GOx@ZIF-8(NiPd) was developed for tandem catalysis. Charge-switchable nanozymes were developed. Site-selective RNA splicing nanozyme was developed. A nanozymes special issue in Progress in Biochemistry and Biophysics was published. Mn3O4 nanozymes with ROS scavenging activities have been developed for in vivo anti-inflammation. A concept entitled "A Step into the Future – Applications of Nanoparticle Enzyme Mimics" was proposed. Facet-dependent oxidase and peroxidase-like activities of Pd nanoparticles were reported. Au@Pt multibranched nanostructures as bifunctional nanozymes were developed. Ferritin coated carbon nanozymes were developed for tumor catalytic therapy. CuO nanozymes were developed to kill bacteria via a light-controlled manner. Enzymatic activity of oxygenated CNT was studied. Nanozymes were used to catalyze the oxidation of l-Tyrosine and l-Phenylalanine to dopachrome. Nanozyme as an emerging alternative to natural enzyme for biosensing and immunoassay was summarized. Standardized assay was proposed for peroxidase-like nanozymes. Semiconductor QDs as nucleases for site-selective photoinduced cleavage of DNA. 2D-MOF nanozyme-based sensor arrays was constructed for detecting phosphates and probing their enzymatic hydrolysis. N-doped carbon nanomaterials as specific peroxidase mimics were reported. Nanozyme sensor arrays were developed to detect analytes from small Molecules to proteins and cells. Copper oxide nanozyme for Parkinson's Disease was reported. Exosome-like nanozyme vesicles for tumor Imaging was developed. A comprehensive review on nanozymes was published by Chemical Society Reviews. A progress report on nanozymes was published. eg occupancy as an effective descriptor was developed for the catalytic activity of perovskite oxide-based peroxidase mimics. A Chemical Reviews on nanozymes was published. A single-atom strategy was used for developing nanozymes. Nanozyme for metal-free bioinspired cascade photocatalysis was reported. A tutorial review on nanozymes was published by Chemical Society Reviews. Cascade nanozyme reactions to convert CO2 into valuable resources was reported. Renal clearable peroxidase-like gold nanoclusters were used for in vivo disease monitoring. Copper/Carbon hybrid nanozyme was developed for antibacterial therapy. A ferritin nanozyme was developed to treat cerebral malaria. A review on nanozymes was published in Acc. Chem. Res. A new strategy called strain effect was developed to modulate the metal nanozyme activity. Prussian blue nanozymes were used to detect hydrogen sulfide (H2S) in the brains of living rats. Photolyase-like CeO2 was reported. An editorial on nanozymes "Can Nanozymes Have an Impact on Sensing?" was published.

2020s

A single-atom nanozyme was developed for sepsis management. Self-assembled single-atom nanozyme was developed for photodynamic therapy treatment of tumor. An ultrasound-switchable nanozyme against multidrug-resistant bacterial infection was reported. A nanozyme-based H₂O₂ homeostasis disruptor for chemodynamic tumor therapy was reported. An iridium oxide nanozyme for cascade reaction was developed for tumor therapy. A book entitled Nanozymology was published. Free radical scavenging nanosponge was engineered for ischemic stroke. A minireview on gold-conjugate based nanozymes. SnSe nanosheets as dehydrogenase mimics were developed. Carbon dot-based topoisomerase I mimic was reported to cleave DNA. Nanozyme sensor arrays were developed to detect pesticides. Bioorthogonal nanozymes were used to treat bacterial biofilms. Rhodium nanozyme was used to treat colon diseases. Fe-N-C nanozyme was developed to study drug-drug interaction. Polymeric nanozyme was developed for second near-infrared photothermal ferrotherapy. Cu5.4O nanozyme was reported for anti-inflammation therapy. CeO2@ZIF-8 nanozyme was developed to treat reperfusion-induced injury in ischemic stroke. Peroxidase-like activity of Fe3O4 was explored to study the electrocatalytic kinetics at single-molecule/single-particle level. Cu-TA nanozyme was fabricated to scavenging ROS from cigarette smoke. Metalloenzyme-like copper nanocluster was reported to have anticancer and imaging activities simultaneously. An integrated nanozyme was developed for anti-inflammation therapy. Enhanced enzyme-like catalytic activity was reported under non-equilibrium conditions for gold nanozymes. A DFT method was proposed to predict the activities of peroxidase-like nanozymes. A hydrolytic nanozyme was developed to construct an immunosensor. An orally administered nanozyme was developed for inflammatory bowel disease therapy. A ligand‐dependent activity engineering strategy was reported to develop glutathione peroxidase‐mimicking MIL‐47(V) metal–organic framework nanozyme for therapy. Single site nanozyme was developed for tumor therapy. SOD-like nanozyme was developed to regulate the mitochondria and neural cell function. Pd12 coordination cage as a photoregulated oxidase-like nanozyme was developed. An NADPH oxidase-like nanozyme was developed. A catalase-like nanozyme was developed for tumor therapy. A defect‐rich adhesive molybdenum disulfide/reduced graphene oxide nanozyme was developed for anti-bacterial. A MOF@COF nanozyme was developed for anti-bacterial. Plasmonic nanozymes were reported. Tumor microenvironment responsive nanozyme was developed for tumor therapy. A protein-engineering inspired method was developed to design highly active nanozymes. An editorial on nanozymes definition was published. A nanozyme therapy for hyperuricemia and ischemic stroke was developed. A perspective on artificial enzymes as well as nanozymes was published by Chemistry World. A review on single atom catalysts including single atom nanozymes was published. Peroxidase-like mixed-FeCo-oxide-based surface-textured nanostructures (MTex) were used for biofilm eradication. A nanozyme with better kinetics than natural peroxidase was developed. A self-protecting nanozyme was developed for Alzheimer's Disease. CuSe nanozymes was developed to treat Parkinson's Disease. A nanocluster-based nanozyme was developed. Glucose oxidase-like gold nanoparticles combined with cyclodextran were used for chiral catalysis. An artificial binuclear copper monooxygenase in a MOF was developed. A review on highly efficient design of nanozymes was published. Ni–Pt peroxidase mimics were developed for bioanalysis. A POM-based nanozyme was reported to protect cells from ROS stress. A gating strategy was used to prepare selective nanozymes. A Mn single atom nanozyme was developed for tumor therapy. A pH-responsive oxidase-like graphitic nanozyme was developed for selective killing of Helicobacter pylori. An engineered FeN3P-centred single-atom nanozyme was developed. Peroxidase- and catalase-like activity of gold nanozymes were modulated. Graphdiyne–cerium oxide nanozymes for radiotherapy of esophageal cancer. Defect engineering was used to develop nanozyme for tumor therapy. A book entitled "Nanozymes for Environmental Engineering" was published. Pd single atom nanozyme was developed for tumor therapy. A HRP-like nanozyme was developed for tumor therapy. The mechanism of GOx-like nanozyme was reported. An Account review on nanozymes was published. A mechanism study on nanonuclease-like nanozyme was reported. A perspective on nanozyme definition was published. Aptananozymes were developed. Ceria nanozyme loaded microneedles helped the hair regrowth. Catalase-like Pt nanozyme was used for small extracellular vesicles analysis. A book on Nanozymes: Advances and Applications was published by CRC Press. A review on nanozyme catalytic turnover was published. A nanozyme was developed for ratiometric molecular imaging. A Fe3O4/Ag/Bi2MoO6 photoactivatable nanozyme was developed for cancer therapy. Co/C as NADH oxidase mimic was reported. Iron oxide nanozyme was used to target biofilms causing tooth decay. A new strategey for high performance nanozyme was developed. A high-throughput computational screening strategy was developed to discover SOD-like nanozymes.

Nanobiotechnology

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Nanobiotechnology 

Nanobiotechnology, bionanotechnology, and nanobiology are terms that refer to the intersection of nanotechnology and biology. Given that the subject is one that has only emerged very recently, bionanotechnology and nanobiotechnology serve as blanket terms for various related technologies.

This discipline helps to indicate the merger of biological research with various fields of nanotechnology. Concepts that are enhanced through nanobiology include: nanodevices (such as biological machines), nanoparticles, and nanoscale phenomena that occurs within the discipline of nanotechnology. This technical approach to biology allows scientists to imagine and create systems that can be used for biological research. Biologically inspired nanotechnology uses biological systems as the inspirations for technologies not yet created. However, as with nanotechnology and biotechnology, bionanotechnology does have many potential ethical issues associated with it.

A ribosome is a biological machine.

The most important objectives that are frequently found in nanobiology involve applying nanotools to relevant medical/biological problems and refining these applications. Developing new tools, such as peptoid nanosheets, for medical and biological purposes is another primary objective in nanotechnology. New nanotools are often made by refining the applications of the nanotools that are already being used. The imaging of native biomolecules, biological membranes, and tissues is also a major topic for nanobiology researchers. Other topics concerning nanobiology include the use of cantilever array sensors and the application of nanophotonics for manipulating molecular processes in living cells.

Recently, the use of microorganisms to synthesize functional nanoparticles has been of great interest. Microorganisms can change the oxidation state of metals. These microbial processes have opened up new opportunities for us to explore novel applications, for example, the biosynthesis of metal nanomaterials. In contrast to chemical and physical methods, microbial processes for synthesizing nanomaterials can be achieved in aqueous phase under gentle and environmentally benign conditions. This approach has become an attractive focus in current green bionanotechnology research towards sustainable development.

Terminology

The terms are often used interchangeably. When a distinction is intended, though, it is based on whether the focus is on applying biological ideas or on studying biology with nanotechnology. Bionanotechnology generally refers to the study of how the goals of nanotechnology can be guided by studying how biological "machines" work and adapting these biological motifs into improving existing nanotechnologies or creating new ones. Nanobiotechnology, on the other hand, refers to the ways that nanotechnology is used to create devices to study biological systems.

In other words, nanobiotechnology is essentially miniaturized biotechnology, whereas bionanotechnology is a specific application of nanotechnology. For example, DNA nanotechnology or cellular engineering would be classified as bionanotechnology because they involve working with biomolecules on the nanoscale. Conversely, many new medical technologies involving nanoparticles as delivery systems or as sensors would be examples of nanobiotechnology since they involve using nanotechnology to advance the goals of biology.

The definitions enumerated above will be utilized whenever a distinction between nanobio and bionano is made in this article. However, given the overlapping usage of the terms in modern parlance, individual technologies may need to be evaluated to determine which term is more fitting. As such, they are best discussed in parallel.

Concepts

Kinesin walking on a microtubule. It is a molecular biological machine that uses protein domain dynamics on nanoscales

Most of the scientific concepts in bionanotechnology are derived from other fields. Biochemical principles that are used to understand the material properties of biological systems are central in bionanotechnology because those same principles are to be used to create new technologies. Material properties and applications studied in bionanoscience include mechanical properties (e.g. deformation, adhesion, failure), electrical/electronic (e.g. electromechanical stimulation, capacitors, energy storage/batteries), optical (e.g. absorption, luminescence, photochemistry), thermal (e.g. thermomutability, thermal management), biological (e.g. how cells interact with nanomaterials, molecular flaws/defects, biosensing, biological mechanisms such as mechanosensation), nanoscience of disease (e.g. genetic disease, cancer, organ/tissue failure), as well as computing (e.g. DNA computing) and agriculture (target delivery of pesticides, hormones and fertilizers. The impact of bionanoscience, achieved through structural and mechanistic analyses of biological processes at nanoscale, is their translation into synthetic and technological applications through nanotechnology.

Nanobiotechnology takes most of its fundamentals from nanotechnology. Most of the devices designed for nano-biotechnological use are directly based on other existing nanotechnologies. Nanobiotechnology is often used to describe the overlapping multidisciplinary activities associated with biosensors, particularly where photonics, chemistry, biology, biophysics, nanomedicine, and engineering converge. Measurement in biology using wave guide techniques, such as dual-polarization interferometry, is another example.

Applications

Applications of bionanotechnology are extremely widespread. Insofar as the distinction holds, nanobiotechnology is much more commonplace in that it simply provides more tools for the study of biology. Bionanotechnology, on the other hand, promises to recreate biological mechanisms and pathways in a form that is useful in other ways.

Nanomedicine

Nanomedicine is a field of medical science whose applications are increasing more and more thanks to nanorobots and biological machines, which constitute a very useful tool to develop this area of knowledge. In the past years, researchers have made many improvements in the different devices and systems required to develop nanorobots. This supposes a new way of treating and dealing with diseases such as cancer; thanks to nanorobots, side effects of chemotherapy have been controlled, reduced and even eliminated, so some years from now, cancer patients will be offered an alternative to treat this disease instead of chemotherapy, which causes secondary effects such as hair loss, fatigue or nausea killing not only cancerous cells but also the healthy ones. At a clinical level, cancer treatment with nanomedicine will consist of the supply of nanorobots to the patient through an injection that will search for cancerous cells while leaving the healthy ones untouched. Patients that will be treated through nanomedicine will not notice the presence of these nanomachines inside them; the only thing that is going to be noticeable is the progressive improvement of their health. Nanobiotechnology is quite important for medicine formulation. It helps a lot in making vaccines as well.

Nanobiotechnology

Nanobiotechnology (sometimes referred to as nanobiology) is best described as helping modern medicine progress from treating symptoms to generating cures and regenerating biological tissues. Three American patients have received whole cultured bladders with the help of doctors who use nanobiology techniques in their practice. Also, it has been demonstrated in animal studies that a uterus can be grown outside the body and then placed in the body in order to produce a baby. Stem cell treatments have been used to fix diseases that are found in the human heart and are in clinical trials in the United States. There is also funding for research into allowing people to have new limbs without having to resort to prosthesis. Artificial proteins might also become available to manufacture without the need for harsh chemicals and expensive machines. It has even been surmised that by the year 2055, computers may be made out of biochemicals and organic salts.

Another example of current nanobiotechnological research involves nanospheres coated with fluorescent polymers. Researchers are seeking to design polymers whose fluorescence is quenched when they encounter specific molecules. Different polymers would detect different metabolites. The polymer-coated spheres could become part of new biological assays, and the technology might someday lead to particles which could be introduced into the human body to track down metabolites associated with tumors and other health problems. Another example, from a different perspective, would be evaluation and therapy at the nanoscopic level, i.e. the treatment of Nanobacteria (25-200 nm sized) as is done by NanoBiotech Pharma.

While nanobiology is in its infancy, there are a lot of promising methods that will rely on nanobiology in the future. Biological systems are inherently nano in scale; nanoscience must merge with biology in order to deliver biomacromolecules and molecular machines that are similar to nature. Controlling and mimicking the devices and processes that are constructed from molecules is a tremendous challenge to face for the converging disciplines of nanobiotechnology. All living things, including humans, can be considered to be nanofoundries. Natural evolution has optimized the "natural" form of nanobiology over millions of years. In the 21st century, humans have developed the technology to artificially tap into nanobiology. This process is best described as "organic merging with synthetic." Colonies of live neurons can live together on a biochip device; according to research from Dr. Gunther Gross at the University of North Texas. Self-assembling nanotubes have the ability to be used as a structural system. They would be composed together with rhodopsins; which would facilitate the optical computing process and help with the storage of biological materials. DNA (as the software for all living things) can be used as a structural proteomic system - a logical component for molecular computing. Ned Seeman - a researcher at New York University - along with other researchers are currently researching concepts that are similar to each other.

Bionanotechnology

DNA nanotechnology is one important example of bionanotechnology. The utilization of the inherent properties of nucleic acids like DNA to create useful materials is a promising area of modern research. Another important area of research involves taking advantage of membrane properties to generate synthetic membranes. Proteins that self-assemble to generate functional materials could be used as a novel approach for the large-scale production of programmable nanomaterials. One example is the development of amyloids found in bacterial biofilms as engineered nanomaterials that can be programmed genetically to have different properties. Protein folding studies provide a third important avenue of research, but one that has been largely inhibited by our inability to predict protein folding with a sufficiently high degree of accuracy. Given the myriad uses that biological systems have for proteins, though, research into understanding protein folding is of high importance and could prove fruitful for bionanotechnology in the future.

Lipid nanotechnology is another major area of research in bionanotechnology, where physico-chemical properties of lipids such as their antifouling and self-assembly is exploited to build nanodevices with applications in medicine and engineering. Lipid nanotechnology approaches can also be used to develop next-generation emulsion methods to maximize both absorption of fat-soluble nutrients and the ability to incorporate them into popular beverages.

Agriculture

In the agriculture industry, engineered nanoparticles have been serving as nano carriers, containing herbicides, chemicals, or genes, which target particular plant parts to release their content. Previously nanocapsules containing herbicides have been reported to effectively penetrate through cuticles and tissues, allowing the slow and constant release of the active substances. Likewise, other literature describes that nano-encapsulated slow release of fertilizers has also become a trend to save fertilizer consumption and to minimize environmental pollution through precision farming. These are only a few examples from numerous research works which might open up exciting opportunities for nanobiotechnology application in agriculture. Also, application of this kind of engineered nanoparticles to plants should be considered the level of amicability before it is employed in agriculture practices. Based on a thorough literature survey, it was understood that there is only limited authentic information available to explain the biological consequence of engineered nanoparticles on treated plants. Certain reports underline the phytotoxicity of various origin of engineered nanoparticles to the plant caused by the subject of concentrations and sizes . At the same time, however, an equal number of studies were reported with a positive outcome of nanoparticles, which facilitate growth promoting nature to treat plant. In particular, compared to other nanoparticles, silver and gold nanoparticles based applications elicited beneficial results on various plant species with less and/or no toxicity. Silver nanoparticles (AgNPs) treated leaves of Asparagus showed the increased content of ascorbate and chlorophyll. Similarly, AgNPs-treated common bean and corn has increased shoot and root length, leaf surface area, chlorophyll, carbohydrate and protein contents reported earlier. The gold nanoparticle has been used to induce growth and seed yield in Brassica juncea.

Tools

This field relies on a variety of research methods, including experimental tools (e.g. imaging, characterization via AFM/optical tweezers etc.), x-ray diffraction based tools, synthesis via self-assembly, characterization of self-assembly (using e.g. MP-SPR, DPI, recombinant DNA methods, etc.), theory (e.g. statistical mechanics, nanomechanics, etc.), as well as computational approaches (bottom-up multi-scale simulation, supercomputing).