This is referred to as cooperative binding. We see cooperativity in large chain molecules made of identical subunits. This is referred to as subunit cooperativity, when a substrate binds to one enzymatic subunit, the rest of the subunits are stimulated and become active. Ligands can either have positive cooperativity, negative cooperativity, or non-cooperativity, an example of positive cooperativity is the binding of oxygen to hemoglobin. One oxygen molecule can bind to the iron of a heme molecule in each of the four chains of a hemoglobin molecule. The oxygen affinity of 3-oxy-hemoglobin is ~300 times greater than that of deoxy-hemoglobin and this behavior leads the affinity curve of hemoglobin to be sigmoidal, rather than hyperbolic as with the monomeric myoglobin. By the same process, the ability for hemoglobin to lose oxygen increases as fewer oxygen molecules are bound, an example of this occurring is the relationship between glyceraldehyde-3-phosphate and the enzyme glyceraldehyde-3-phosphate dehydrogenase.
Homotropic cooperativity refers to the fact that the causing the cooperativity is the one that will be affected by it. Heterotropic cooperativity is where a third party substance causes the change in affinity, for example, unwinding of DNA involves cooperativity, Portions of DNA must unwind in order for DNA to carry out replication and recombination. The cooperative unit size is the number of adjacent bases that tend to unwind as a unit due to the effects of positive cooperativity. This phenomenon applies to other types of molecules as well, such as the folding and unfolding of proteins. Subunit cooperativity is measured on the relative scale known as Hills Constant, a simple and widely used model for molecular interactions is the Hill Equation. This provides a way to quantify cooperative binding by describing the fraction of saturated ligand binding sites as a function of the ligand concentration, in all of the above types of cooperativity, entropy plays a role. For example, in the case of oxygen binding to hemoglobin and this represents a state of higher entropy compared to a fourth oxygen having one available binding site.
Thus, in transition from the unbound to the bound state, the first oxygen must overcome a larger entropy change than the last oxygen in order to bind to the hemoglobin
A cofactor is a non-protein chemical compound or metallic ion that is required for a proteins biological activity to happen. These proteins are enzymes, and cofactors can be considered helper molecules that assist in biochemical transformations. A coenzyme that is tightly or even covalently bound is termed a prosthetic group, the two subcategories under coenzyme are cosubstrates and prosthetic groups. Cosubstrates are transiently bound to the protein and will be released at some point, the prosthetic groups, on the other hand, are bound permanently to the protein. Both of them have the function, which is to facilitate the reaction of enzymes. Additionally, some sources limit the use of the cofactor to inorganic substances. An inactive enzyme without the cofactor is called an apoenzyme, while the enzyme with cofactor is called a holoenzyme. Some enzymes or enzyme complexes require several cofactors, organic cofactors are often vitamins or made from vitamins. Many contain the nucleotide adenosine monophosphate as part of their structures, such as ATP, coenzyme A, FAD and this common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world.
It has been suggested that the AMP part of the molecule can be considered to be a kind of handle by which the enzyme can grasp the coenzyme to switch it between different catalytic centers. Cofactors can be divided into two groups, organic cofactors, such as flavin or heme, and inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+. Organic cofactors are sometimes divided into coenzymes and prosthetic groups. The term coenzyme refers specifically to enzymes and, as such, on the other hand, prosthetic group emphasizes the nature of the binding of a cofactor to a protein and, refers to a structural property. Different sources give different definitions of coenzymes, cofactors. It should be noted that terms are often used loosely. However, the author could not arrive at a single all-encompassing definition of a coenzyme, the study of these cofactors falls under the area of bioinorganic chemistry. In nutrition, the list of essential trace elements reflects their role as cofactors, in humans this list commonly includes iron, manganese, copper and molybdenum.
Although chromium deficiency causes impaired glucose tolerance, no human enzyme that uses this metal as a cofactor has been identified, iodine is an essential trace element, but this element is used as part of the structure of thyroid hormones rather than as an enzyme cofactor
Enzyme promiscuity is the ability of an enzyme to catalyse a fortuitous side reaction in addition to its main reaction. Although enzymes are remarkably specific catalysts, they can often perform side reactions in addition to their main and these promiscuous activities are usually slow relative to the main activity and are under neutral selection. An example of this is the atrazine chlorohydrolase from Pseudomonas sp, ADP which evolved from melamine deaminase, which has very small promiscuous activity towards atrazine, a man-made chemical. Enzymes are evolved to catalyse a reaction on a particular substrate with a high catalytic efficiency. Several theoretical models exist to predict the order of duplication and specialisation events, on the other, enzymes may evolve an increased secondary activity with little loss to the primary activity with little adaptive conflict. A study of three distinct hydrolases has shown the main activity is robust towards change, whereas the activities are more plastic.
Specifically, selecting for an activity that is not the activity, does not initially diminish the main activity. The most recent and most clear cut example of evolution is the rise of bioremediating enzymes in the past 60 years. Due to the low number of amino acid changes, these provide an excellent model to investigate enzyme evolution in nature. This issue can be resolved thanks to ancestral reconstruction and this variability in ancestral specificity has not only been observed between different genes, but within the same gene family. Antithetically, the ancestor before the split had a more pronounced isomaltose-like glucosidase activity. Roy Jensen in 1976 theorised that primordial enzymes had to be highly promiscuous in order for networks to assemble in a patchwork fashion. This primordial catalytic versatility was lost in favour of highly catalytic specialised orthologous enzymes, as a consequence, many central-metabolic enzymes have structural homologues that diverged before the last universal common ancestor.
Promiscuity is however not only a primordial trait, in fact it is very widespread property in modern genomes, a series of experiments have been conducted to assess the distribution of promiscuous enzyme activities in E. coli. In E. coli 21 out of 104 single-gene knockouts tested could be rescued by overexpressing a noncognate E. coli protein, overexpressing the ORF collection allowed E. coli to gain over an order of magnitude in resistance in 86 out 237 toxic environment. Homologues are sometimes known to display promiscuity towards each others main reactions, despite the divergence the homologues have a varying degree of reciprocal promiscuity, the differences in promiscuity are due to mechanisms involved, particularly the intermediate required. Examples of these are enzymes for primary and secondary metabolism in plants, a promiscuous activity is a non-native activity the enzyme did not evolve to do, but arises due to an accommodating conformation of the active site. When the specificity of enzyme was probed, it was found that it was selective against natural amino acids that were not phenylalanine
Isomerases are a general class of enzymes which convert a molecule from one isomer to another. Isomerases can either facilitate intramolecular rearrangements in which bonds are broken, the general form of such a reaction is as follows, A–B → B–A There is only one substrate yielding one product. This product has the molecular formula as the substrate but differs in bond connectivity or spatial arrangements. Isomerases catalyze reactions across many biological processes, such as in glycolysis, Isomerases catalyze changes within one molecule. They convert one isomer to another, meaning that the end product has the molecular formula. Isomers themselves exist in many varieties but can generally be classified as structural isomers or stereoisomers, structural isomers have a different ordering of bonds and/or different bond connectivity from one another, as in the case of hexane and its four other isomeric forms. Stereoisomers have the same ordering of bonds and the same connectivity. For example, 2-butene exists in two forms, cis-2-butene and trans-2-butene.
The sub-categories of isomerases containing racemases and cis-trans isomers are examples of enzymes catalyzing the interconversion of stereoisomers, intramolecular lyases and transferases catalyze the interconversion of structural isomers. The prevalence of each isomer in nature depends in part on the isomerization energy, isomers close in energy can interconvert easily and are often seen in comparable proportions. Isomerases can increase the rate by lowering the isomerization energy. Calculating isomerase kinetics from experimental data can be more difficult than for other enzymes because the use of product inhibition experiments is impractical, There are practical difficulties in determining the rate-determining step at high concentrations in a single isomerization. Instead, tracer perturbation can overcome these technical difficulties if there are two forms of the unbound enzyme and this technique uses isotope exchange to measure indirectly the interconversion of the free enzyme between its two forms.
The radiolabeled substrate and product diffuse in a time-dependent manner, when the system reaches equilibrium the addition of unlabeled substrate perturbs or unbalances it. As equilibrium is established again, the substrate and product are tracked to determine energetic information. This technique was adopted to study the profile of proline racemase. Generally, the names of isomerases are formed as substrate isomerase, enzyme-catalyzed reactions each have a uniquely assigned classification number. Isomerase-catalyzed reactions have their own EC category, EC5, Isomerases are further classified into six subclasses, This category includes and epimerases)
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 residues that form bonds with the substrate. The active site is usually a groove or pocket of the enzyme which can be located in a tunnel within the enzyme. An active site can catalyse a reaction repeatedly as its residues are not altered at the end of the reaction, usually, an enzyme molecule has only one active site, and the active site fits with one specific type of substrate. An active site contains a site that binds the substrate. Residues in the site form hydrogen bonds, hydrophobic interactions. In order to function, the site needs to be in a specific conformation. A tighter fit between a site and the substrate molecule is believed to increase efficiency of a reaction. Most enzymes have deeply buried active sites, which can be accessed by a substrate via access channels, there are two proposed models of how enzymes fit to their specific substrate, the lock and key model and the induced fit model.
Emil Fischers lock and key model assumes that the site is a perfect fit for a specific substrate. Daniel Koshlands theory of enzyme-substrate binding is that the active site, the induced fit model is a development of the lock-and-key model and assumes that an active site is flexible and it changes shape until the substrate is completely bound. The substrate is thought to induce a change in the shape of the active site, the hypothesis predicts that the presence of certain residues in the active site will encourage the enzyme to locate the correct substrate. Conformational changes may occur as the substrate is bound. After the products of the move away from the enzyme. Once the substrate is bound and oriented in the active site, 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 energy of a reaction. They do this by a number of different mechanisms, they can act as donors or acceptors of protons or other groups on the substrate to facilitate the reaction.
They can form electrostatic interactions to stabilise charge buildup on the state or leaving group
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished, mass concentration, molar concentration, number concentration, the term concentration can be applied to any kind of chemical mixture, but most frequently it refers to solutes and solvents in solutions. The molar concentration has variants such as concentration and osmotic concentration. To concentrate a solution, one must add more solute, or reduce the amount of solvent, by contrast, to dilute a solution, one must add more solvent, or reduce the amount of solute. Unless two substances are fully miscible there exists a concentration at which no further solute will dissolve in a solution, at this point, the solution is said to be saturated. If additional solute is added to a solution, it will not dissolve, except in certain circumstances. Instead, phase separation will occur, leading to coexisting phases, the point of saturation depends on many variables such as ambient temperature and the precise chemical nature of the solvent and solute.
Concentrations are often called levels, reflecting the mental schema of levels on the axis of a graph. There are four quantities that describe concentration, The mass concentration ρ i is defined as the mass of a constituent m i divided by the volume of the mixture V, ρ i = m i V. The molar concentration c i is defined as the amount of a constituent n i divided by the volume of the mixture V, c i = n i V, more commonly the unit mol/L is used. The number concentration C i is defined as the number of entities of a constituent N i in a divided by the volume of the mixture V, C i = N i V. The volume concentration ϕ i is defined as the volume of a constituent V i divided by the volume of the mixture V, ϕ i = V i V. Being dimensionless, it is expressed as a number, e. g.0.18 or 18%, several other quantities can be used to describe the composition of a mixture. Note that these should not be called concentrations, normality is defined as the molar concentration c i divided by an equivalence factor f e q.
Since the definition of the equivalence factor depends on context, IUPAC, the SI unit for molality is mol/kg. The mole fraction x i is defined as the amount of a constituent n i divided by the amount of all constituents in a mixture n t o t, x i = n i n t o t. However, the deprecated parts-per notation is used to describe small mole fractions. The mole ratio r i is defined as the amount of a constituent n i divided by the amount of all other constituents in a mixture
As such, these points satisfy x =0. If the curve in question is given as y = f, functions which are undefined at x =0 have no y-intercept. If the function is linear and is expressed in slope-intercept form as f = a + b x, some 2-dimensional mathematical relationships such as circles and hyperbolas can have more than one y-intercept. Because functions associate x values to no more than one y value as part of their definition, analogously, an x-intercept is a point where the graph of a function or relation intersects with the x-axis. As such, these points satisfy y=0, the zeros, or roots, of such a function or relation are the x-coordinates of these x-intercepts. Unlike y-intercepts, functions of the form y = f may contain multiple x-intercepts, the x-intercepts of functions, if any exist, are often more difficult to locate than the y-intercept, as finding the y intercept involves simply evaluating the function at x=0. The notion may be extended for 3-dimensional space and higher dimensions, as well as for other coordinate axes, for example, one may speak of the I-intercept of the current-voltage characteristic of, say, a diode
Diffusion limited enzyme
A Diffusion limited enzyme is an enzyme which catalyses a reaction so efficiently that the rate limiting step is that of substrate diffusion into the active site, or product diffusion out. This is known as kinetic perfection or catalytic perfection, since the rate of catalysis of such enzymes is set by the diffusion-controlled reaction, it therefore represents an intrinsic, physical constraint on evolution. Diffusion limited perfect enzymes are very rare, most enzymes catalyse their reactions to a rate that is 1, 000-10,000 times slower than this limit. This is due to both the limitations of difficult reactions, and the evolutionary limitations that such high reaction rates do not confer any extra fitness. The theory of diffusion-controlled reaction was utilized by R. A. Alberty, Gordon Hammes, and Manfred Eigen to estimate the upper limit of enzyme-substrate reaction, according to their estimation, the upper limit of enzyme-substrate reaction was 109 M−1 s−1. To address such a paradox, the new upper limit found by Chou et al. for enzyme-substrate reaction was further discussed and analyzed by a series of follow-up studies.
Kinetically perfect enzymes have a specificity constant, kcat/Km, on the order of 108 to 109 M−1 s−1, the rate of the enzyme-catalysed reaction is limited by diffusion and so the enzyme processes the substrate well before it encounters another molecule. Some enzymes operate with kinetics which are faster than diffusion rates, several mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by drawing their substrate in, some invoke a quantum-mechanical tunneling explanation whereby a proton or an electron can tunnel through activation barriers, although proton tunneling remains a somewhat controversial idea. It is worth noting that there are not many kinetically perfect enzymes and this can be explained in terms of natural selection. An increase in speed may be favoured as it could confer some advantage to the organism. However, when the catalytic speed outstrips diffusion speed there is no advantage to increase the speed even further. The diffusion limit represents a physical constraint on evolution.
Increasing the catalytic speed past the speed will not aid the organism in any way
An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. Since blocking an enzymes activity can kill a pathogen or correct a metabolic imbalance and they are used in pesticides. The binding of an inhibitor can stop a substrate from entering the active site and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically and these inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind to the enzyme, many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in biochemistry and pharmacology. A medicinal enzyme inhibitor is often judged by its specificity and its potency, a high specificity and potency ensure that a drug will have few side effects and thus low toxicity.
Enzyme inhibitors occur naturally and are involved in the regulation of metabolism, for example, enzymes in a metabolic pathway can be inhibited by downstream products. This type of negative feedback slows the production line when products begin to build up and is an important way to maintain homeostasis in a cell, other cellular enzyme inhibitors are proteins that specifically bind to and inhibit an enzyme target. This can help control enzymes that may be damaging to a cell, a well-characterised example of this is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions. Natural enzyme inhibitors can be poisons and are used as defences against predators or as ways of killing prey, reversible inhibitors attach to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding, in contrast to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.
There are four kinds of reversible enzyme inhibitors and they are classified according to the effect of varying the concentration of the enzymes substrate on the inhibitor. In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the right. This usually results from the inhibitor having an affinity for the site of an enzyme where the substrate binds. This type of inhibition can be overcome by high concentrations of substrate. However, the apparent Km will increase as it takes a higher concentration of the substrate to reach the Km point, competitive inhibitors are often similar in structure to the real substrate. In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex and this type of inhibition causes Vmax to decrease and Km to decrease
A protein superfamily is the largest grouping of proteins for which common ancestry can be inferred. Usually this common ancestry is inferred from structural alignment and mechanistic similarity, sequence homology can be deduced even if not apparent. Superfamilies typically contain several protein families which show sequence similarity within each family, the term protein clan is commonly used for protease superfamilies based on the MEROPS protease classification system. Superfamilies of proteins are identified using a number of methods, closely related members can be identified by different methods to those needed to group the most evolutionarily divergent members. Historically, the similarity of different amino acid sequences has been the most common method of inferring homology, amino acid sequence is typically more conserved than DNA sequence, so is a more sensitive detection method. Since some of the amino acids have similar properties, conservative mutations that interchange them are often neutral to function, the most conserved sequence regions of a protein often correspond to functionally important regions like catalytic sites and binding sites, since these regions are less tolerant to sequence changes.
Using sequence similarity to infer homology has several limitations, there is no minimum level of sequence similarity guaranteed to produce identical structures. Over long periods of evolution, related proteins may show no sequence similarity to one another. Sequences with many insertions and deletions can sometimes be difficult to align, in the PA clan of proteases, for example, not a single residue is conserved through the superfamily, not even those in the catalytic triad. Conversely, the families that make up a superfamily are defined on the basis of their sequence alignment. Nevertheless, sequence similarity is the most commonly used form of evidence to infer relatedness, in the absence of structural information, sequence similarity constrains the limits of which proteins can be assigned to a superfamily. Structure is much more conserved than sequence, such that proteins with highly similar structures can have entirely different sequences. Over very long timescales, very few residues show detectable amino acid sequence conservation, however secondary structural elements.
Conformational changes of the structure may be conserved, as is seen in the serpin superfamily. Consequently, protein structure can be used to detect homology between proteins even when no evidence of relatedness remains in their sequences. Structural alignment programs, such as DALI, use the 3D structure of a protein of interest to find proteins with similar folds, however, on rare occasions, related proteins may evolve to be structurally dissimilar and relatedness can only be inferred by other methods. The catalytic mechanism of enzymes within a superfamily is typically conserved, catalytic residues tend to occur in the same order in the protein sequence. However, mechanism alone is not sufficient to infer relatedness, since some catalytic mechanisms have been convergently evolved multiple times independently, protein superfamilies represent the current limits of our ability to identify common ancestry
A catalytic triad refers to the three amino acid residues that function together at the centre of the active site of some hydrolase and transferase enzymes. An Acid-Base-Nucleophile triad is a motif for generating a nucleophilic residue for covalent catalysis. The nucleophile is most commonly a serine or cysteine amino acid, as well as divergent evolution of function, catalytic triads show some of the best examples of convergent evolution. Chemical constraints on catalysis have led to the same solution independently evolving in at least 23 separate superfamilies. Their mechanism of action is one of the best studied in biochemistry. The enzymes trypsin and chymotrypsin were first purified in the 1930s, a serine in each of trypsin and chymotrypsin was identified as the catalytic nucleophile in the 1950s. The structure of chymotrypsin was solved by X-ray crystallography in the 1960s, other proteases were sequenced and aligned to reveal a family of related proteases, now called the S1 family. Simultaneously, the structures of the evolutionarily unrelated papain and subtilisin proteases were found to contain analogous triads, the charge-relay mechanism for the activation of the nucleophile by the other triad members was proposed in the late 1960s.
As more protease structures were solved by X-ray crystallography in the 1970s and 80s, understanding how chemical constraints on evolution led to the convergence of so many enzyme families on the same triad geometries has developed in the 2010s. The massive body of work on the charge-relay, covalent catalysis used by catalytic triads has led to the mechanism being the best characterised in all of biochemistry. Enzymes that contain an catalytic triad use it for one of two types, either to split a substrate or to transfer one portion of a substrate over to a second substrate. Triads are an inter-dependent set of residues in the site of an enzyme. These triad residues act together to make the nucleophile member highly reactive, catalytic triads perform covalent catalysis using a residue as a nucleophile. The reactivity of the residue is increased by the functional groups of the other triad members. The nucleophile is polarised and oriented by the base, which is itself bound, catalysis is performed in two stages.
First, the nucleophile attacks the carbonyl carbon and forces the carbonyl oxygen to accept an electron. The build-up of negative charge on this intermediate is stabilized by an oxanion hole within the active site. The intermediate collapses back to a carbonyl, ejecting the first half of the substrate, the ejection of this first leaving group is often aided by donation of a proton by the base