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Regulatory enzymes , which facilitate the transferring of phosphate groups to the specific substrates, are called kinases. Protein kinases are the numerous groups of kinases, which involve in the regulation of cell function and further modification. In prokaryotes and eukaryotes, several protein kinases exert specific and reversible control on protein phosphorylation. Protein kinases are classified both by the type of amino acid they phosphorylate in the protein target and by their location in the cell. Biochemically, phosphorylation distinguishes five different groups, that is, histidine, serine, threonine or tyrosine, and dual-specificity kinases. The majority of eukaryotic kinases are serine/threonine-specific protein kinases. Tyrosine-specific protein kinases are used in signal transduction processes in eukaryotes and a low level of activity is detectable in yeast. Protein kinases are established in cytosol and in the plasma membrane. Ser/Thr protein kinases and the dual-specificity kinases are most often cytosolic. Most tyrosine kinases are found on the plasma membrane. Protein kinases have profound effects on a cell with highly regulated activity by turning on and off and by binding of proteins with advantage as regulatory strategy. Regulation of protein kinases is ubiquitous in eukaryotic development, physiology, and metabolism and deregulation is a common cause of disease, particularly cancer.
Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitylated proteins by the proteasome, but other functions of ubiquitin have been discovered that do not involve the proteasome. Some proteins are modified by a single ubiquitin or short ubiquitin chains. Instead of sending proteins to their death through the proteasome, monoubiquitylation regulates processes that range from membrane transport to transcriptional regulation.
The CDK6 gene is conserved in eukaryotes, including the budding yeast and the nematode Caenorhabditis elegans.  The CDK6 gene is located on chromosome 7 in humans. The gene spans 231,706 base pairs and encodes a 326 amino acid protein with a kinase function.  The gene is overexpressed in cancers like lymphoma, leukemia, medulloblastoma and melanoma associated with chromosomal rearrangements.  The CDK6 protein contains a catalytic core composed of a serine/threonine domain.  This protein also contains an ATP-binding pocket, inhibitory and activating phosphorylation sites, a PSTAIRE-like cyclin-binding domain and an activating T-loop motif.  After binding the Cyclin in the PSTAIRE helix, the protein changes its conformational structure to expose the phosphorylation motif.  The protein can be found in the cytoplasm and the nucleus, however most of the active complexes are found in the nucleus of proliferating cells. 
Cell cycle Edit
In 1994, Matthew Meyerson and Ed Harlow investigated the product of a close analogous gene of CDK4.  This gene, identified as PLSTIRE was translated into a protein that interacted with the cyclins CD1, CD2 and CD3 (same as CDK4), but that was different from CDK4 the protein was then renamed CDK6 for simplicity.  In mammalian cells, cell cycle is activated by CDK6 in the early G1 phase  through interactions with cyclins D1, D2 and D3.  There are many changes in gene expression that are regulated through this enzyme.  After the complex is formed, the C-CDK6 enzymatic complex phosphorylates the protein pRb.  After its phosphorylation, pRb releases its binding partner E2F, a transcriptional activator, which in turn activates DNA replication.  The CDK6 complex ensures a point of switch to commit to division responding to external signals, like mitogens and growth factors. 
CDK6 is involved in a positive feedback loop that activates transcription factors through a reaction cascade.  Importantly, these C-CDK complexes act as a kinase, phosphorylating and inactivating the protein of Rb and p-Rb related “pocket proteins” p107 and p130.  While doing this, the CDK6 in conjunction with CDK4, act as a switch signal that first appears in G1,  directing the cell towards S phase of the cell cycle. 
CDK6 is important for the control of G1 to S phase transition.  However, in recent years, new evidence proved that the presence of CDK6 is not essential for proliferation in every cell type,  the cell cycle has a complex circuitry of regulation and the role of CDK6 might be more important in certain cell types than in others, where CDK4 or CDK2 can act as protein kinases compensating its role.  
Cellular development Edit
In mutant Knockout mice of CDK6, the hematopoietic function is impaired, regardless of otherwise organism normal development.  This might hint additional roles of CDK6 in the development of blood components.  There are additional functions of CDK6 not associated with its kinase activity.  For example, CDK6 is involved in the differentiation of T cells, acting as an inhibitor of differentiation.  Even though CDK6 and CDK4 share 71% amino acid identity, this role in differentiation is unique to CDK6.  CDK6 has also been found to be important in the development of other cell lines, for example, CDK6 has a role in the alteration of the morphology of astrocytes  and in the development of other stem cells.  
DNA protection Edit
CDK6 differs from CDK4 in other important roles.  For example, CDK6 plays a role in the accumulation of the apoptosis proteins p53 and p130, this accumulation keeps cells from entering cell division if there is DNA damage, activating pro- apoptotic pathways. 
Metabolic homeostasis Edit
Studies in the metabolic control of cells have revealed yet another role of CDK6.  This new role is associated with the balance of the oxidative and non-oxidative branches of the pentose pathway in cells.  This pathway is a known route altered in cancer cells, when there is an aberrant overexpression of CDK6 and CDK4.  The overexpression of these proteins provides the cancer cells with a new hallmark capability of cancer the deregulation of the cell metabolism. 
Centrosome stability Edit
In 2013, researchers discovered yet another role of CDK6.  There is evidence that CDK6 associates with the centrosome and controls organized division and cell cycle phases in neuron production.  When the CDK6 gene is mutated in these developing lines, the centrosomes are not properly divided, this could lead to division problems such as aneuploidy, which in turns leads to health issues like primary microcephaly. 
CDK6 is positively regulated primarily by its union to the D cyclins D1, D2 and D3. If this subunit of the complex is not available, CDK6 is not active or available to phosphorylate the pRb substrate.  An additional positive activator needed by CDK6 is the phosphorylation in a conserved threonine residue located in 177 position, this phosphorylation is done by the cdk-activating kinases, CAK.  Additionally, CDK6 can be phosphorylated and activated by the Kaposi's sarcoma-associated herpes virus, stimulating the CDK6 over activation and uncontrolled cell proliferation. 
CDK6 is negatively regulated by binding to certain inhibitors that can be classified in two groups  CKIs or CIP/KIP family members like the protein p21  and p27 act blocking and inhibiting the assembled C-CDKs binding complex enzymes  in their catalytic domain. 
Furthermore, inhibitors of the INK4 family members like p15, p16, p18 and p19 inhibit the monomer of CDK6, preventing the complex formation.  
CDK6 is a protein kinase activating cell proliferation, it is involved in an important point of restriction in the cell cycle.  For this reason, CDK6 and other regulators of the G1 phase of the cell cycle are known to be unbalanced in more than 80-90% of tumors.  In cervical cancer cells, CDK6 function has been shown to be altered indirectly by the p16 inhibitor.  CDK6 is also overexpressed in tumors that exhibit drug resistance, for example glioma malignancies exhibit resistance to chemotherapy using temozolomide (TMZ) when they have a mutation overexpressing CDK6.  Likewise, the overexpression of CDK6 is also associated with resistance to hormone therapy using the anti oestrogen Fluvestrant in breast cancer. 
Loss of normal cell cycle control is the first step to developing different hallmarks of cancer alterations of CDK6 can directly or indirectly affect the following hallmarks disregulated cell cellular energetics, sustaining of proliferative signaling, evading growth suppressors and inducing angiogenesis,  for example, deregulation of CDK6 has been shown to be important in lymphoid malignancies by increasing angiogenesis, a hallmark of cancer.  These features are reached through upregulation of CDK6 due to chromosome alterations or epigenetic dysregulations.  Additionally, CDK6 might be altered through genomic instability, a mechanism of downregulation of tumor suppressor genes this represents another evolving hallmark of cancer. 
Medulloblastoma is the most common cause of brain cancer in children.  About a third of these cancers have upregulated CDK6, representing a marker for poor prognosis for this disease.  Since it is so common for these cells to have alterations in CDK6, researchers are seeking for ways to downregulate CDK6 expression acting specifically in those cell lines. The MicroRNA (miR) -124 has successfully controlled cancer progression in an in-vitro setting for medulloblastoma and glioblastoma cells.  Furthermore, researchers have found that it successfully reduces the growth of xenograft tumors in rat models. 
As a drug target Edit
The direct targeting of CDK6 and CDK4 should be used with caution in the treatment of cancer, because these enzymes are important for the cell cycle of normal cells as well.  Furthermore, small molecules targeting these proteins might increase drug resistance events.  However, these kinases have been shown to be useful as coadjuvants in breast cancer chemotherapy.  Another indirect mechanism for the control of CDK6 expression, is the use of a mutated D-cyclin that binds with high affinity to CDK6, but does not induce its kinase activity.  this mechanism was studied in the development of mammary tumorigenesis in rat cells, however, the clinical effects have not yet been shown in human patients.  A
MP4. Regulation of Metabolic Pathways: How Is It Regulated?
- Contributed by Chris Schaller
- Professor (Chemistry) at College of Saint Benedict/Saint John's University
Exquisite mechanisms have evolved that control the flux of metabolites through metabolic pathways to insure that the output of the pathways meets biological demand and that energy in the form of ATP is not wasted by having opposing pathways run concomitantly in the same cell.
Enzymes can be regulated by changing the activity of a preexisting enzyme or changing the amount of an enzyme.
A. Changing the activity of a pre-existing enzyme: The quickest way to modulate the activity of an enzyme is to alter the activity of an enzyme that already exists in the cell. The list below, illustrated in the following figure, gives common ways to regulate enzyme activity
- Substrate availability: Substrates (reactants) bind to enzymes with a characteristic affinity (characterized by a dissociation constant) and a kinetic parameter called Km (units of molarity). If the actual concentration of a substrate in a cell is much less than the Km, the activity of the enzyme is very low. If the substrate concentration is much greater than Km, the enzyme active site is saturated with substrate and the enzyme is maximally active.
- Product inhibition: A product of an enzyme-catalyzed reaction often resembles a starting reactant, so it should be clear that the product should also bind to the activity site, albeit probably with lower affinity. Under conditions in which the product of a reaction is present in high concentration, it would be energetically advantageous to the cell if no more product was synthesized. Product inhibition is hence commonly observed. Likewise it be energetically advantageous to a cell if the end product of an entire pathway could likewise bind to the initial enzyme in the pathways and inhibit it, allowing the whole pathway to be inhibited. This type of feedback inhibition is commonly oberved
- Allosteric regulation: As many pathways are interconnected, it would be optimal if the molecules of one pathway affected the activity of enzymes in another interconnected pathway, even if the molecules in the first pathway are structurally dissimilar to reactants or products in a second pathway. Molecules that bind to sites on target enzymes other than the active site (allosteric sites) can regulate the activity of the target enzyme. These molecules can be structurally dissimilar to those that bind at the active site. They do so my conformational changes which can either activate or inhibit the target enzyme's activity.
- pH and enzyme conformation: Changes in pH which can accompany metabolic process such as respiration (aerobic glycolysis for example) can alter the conformation of an enzyme and hence enzyme activity. The initial changes are covalent (change in protonation state of the protein) which can lead to an alteration in the delicate balance of forces that affect protein structure.
- pH and active site protonation state: Changes in pH can affect the protonation state of key amino acid side chains in the active site of proteins without affecting the local or global conformation of the protein. Catalysis may be affected if the mechanism of catalysis involves an active site nucleophile (for example), that must be deprotonated for activity.
- Covalent modification: Many if not most proteins are subjected to post-translational modifications which can affect enzyme activity through local or global shape changes, by promoting or inhibiting binding interaction of substrates and allosteric regulators, and even by changing the location of the protein within the cell. Proteins may be phosphorylated, acetylated, methylated, sulfated, glycosylated, amidated, hydroxylated, prenylated, myristolated, often in a reversible fashion. Some of these modifications are reversible. Regulation by phosphorylation through the action of kinases, and dephosphorylation by phosphates is extremely common. Control of phosphorylation state is mediated through signal transduction process starting at the cell membrane, leading to the activation or inhibition of protein kinases and phosphatases within the cell.
Figure: Regulation of the Activity of Pre-existing Enzymes
Extracellular regulated kinase 2 (ERK2), also known as mitogen activate protein kinase 2 (MAPK2) is a protein the plays a vital role in cell signaling across the cell membrane. Phosphoryation of ERK2 on Threonine 183 (Thr153) and Tyrosine 185 (Tyr185) leads to a structural change in the protein and the regulation of its activity.
B. Changing the amount of an enzyme: Another and less immediate but longer duration method to modulate the activity of an enzyme is to alter the activity of an enzyme that already exists in the cell. The list below, illustrated in the following figure, shows way in which enzyme concentration is regulated.
- Alternation in transcription of enzyme's gene: Extracellular signal (hormones, neurotransmitters, etc) can lead to signal transductions responses and ultimate activation or inhibition of the transcription of the gene for a protein enzyme. These changes result from recruitment of transcription factors (proteins) to DNA sequences that regulate transcription of the enzyme gene.
- Degradation of messenger RNA for the enzyme: The levels of messenger RNA for a protein will directly determin the amount of that protein synthesized. Small inhibitor RNAs, derived from microRNA molecules transcribed from cellular DNA, can bind to specific sequences in the mRNA of a target enzyme. The resulting double-stranded RNA complex recruits an enzyme (Dicer) that cleaves the complex with the effect of decreasing translation of the protein enzyme from its mRNA.
- Co/Post translational changes: Once a protein enzymes is translated from its mRNA, it can undergo a changes to affect enzyme levels. Some proteins are synthesized in a "pre"form which must be cleaved in a targeted and limited fashion by proteases to active the protein enzyme. Some proteins are not fully folded and must bind to other factors in the cell to adopted a catalytically active form. Finally, fully active protein can be fully proteolyzed by the proteasome, a complex within cells, or in lysosomes, which are organelles within cells containing proteolytic enzymes.
Next we will consider which enzymes in pathways make the best target for regulation.
Antibody Arrays in Biomarker Discovery
Jarad J. Wilson , . Ruo-Pan Huang , in Advances in Clinical Chemistry , 2015
1.5.1 Protein expression
First, protein expression levels provide unique insight into the existence of whole proteins in a system as they apply to marking a biological state. Alterations in normal levels of protein(s) may indicate that the system has gone awry. Levels of a protein may go up or down in response to a particular biological state or disease. C-reactive protein (CRP) is a good example of a key biomarker upregulated during, or in response to, inflammation. It is synthesized in the liver in response to factors secreted by both macrophages and adipocytes during an inflammatory response. CRP is particularly valuable as a biomarker as it gages the status of an inflammatory response which could be driven by countless diseases or medical disorders  .
Synthesis of mevalonate from acetyl-CoA
The first stage of cholesterol synthesis leads to the production of the intermediate mevalonate. The synthesis of mevalonate is the committed, rate-limiting step in cholesterol formation. In this reaction, two molecules of acetyl-CoA condense, forming acetoacetyl-CoA, which then condenses with a third molecule of acetyl-CoA to yield the six-carbon compound (eta)-hydroxy-(eta)-methylglutaryl-CoA (HMG-CoA) (Figure 6.3), (the cytosolic HMG-CoA synthase in this reaction is distinct from the mitochondrial HMG-CoA synthase that catalyses a similar reaction involved in production of ketone bodies.). The committed step and major point of regulation of cholesterol synthesis involves reduction of HMG-CoA to mevalonate, in a reaction that is catalyzed by HMG-CoA reductase.
Figure 6.3: Regulatory step catalyzed by HMG-CoA reductase.
The subsequent steps of the pathway proceed largely unregulated and mevalonate is used to synthesize isoprenoid units (5-carbon units). These 5- carbon chains are joined in a head to tail fashion generating squalene, 30- carbons, which undergoes a cyclization reaction after epoxidation. The cyclized product, lanosterol, undergoes several reactions to generate the final product, cholesterol.
BACKGROUND THEORY AND PRE-LABORATORY PREPARATION
The metabolism of nutrients is a tightly regulated process in which different types of tissues, cells, organelles, and biomolecules participate in the coordination and regulation of metabolism. In higher animals, the activity of the regulating enzyme of a biochemical pathway may be regulated by short- and long-term mechanisms. Depending on physiological needs, the genetic control of enzyme synthesis may regulate the amount of enzyme available by a long-term mechanism [ 6 – 8 ].
The enzymes involved in the metabolism of protein, carbohydrates, and lipids may also be regulated by short-term mechanism. One of these mechanism is the allosteric regulation, where the binding of small molecules, which may be an activator, inhibitor, or substrate (modulators), increases or decreases enzyme activity. Thus, enzyme activity can be regulated according to physiological needs that may arise during the cell life [ 6 , 7 ].
Enzyme activities can also be regulated by reversible covalent modifications, such as the addition of phosphate groups to serine, threonine, or tyrosine residues. Covalent modification is a highly efficient pathway to control not only enzyme in metabolic reactions, such as glycogen synthesis and break down, but also many other cellular functions, including cell growth and differentiation [ 6 – 8 ].
Another important mechanism to regulate protein function is the proteolytic activation that can act like switches, turning proteins “on” and “off.” The proteolytic activation is a special form of covalent modification in which the inactive precursor of the enzyme is activated by an irreversible cleavage. Enzymes active in digestive and blood clotting processes are switched on by proteolytic cleavage. In general, these proenzymes are synthesized as slightly longer polypeptide chains than true active enzymes, and usually they are part of a cascade of mechanisms. As observed in Fig. 1, after secretion in the duodenum, trypsinogen is converted to trypsin by enteropeptidase, which hydrolyzes a unique lysine-isoleucine peptide bond in trypsinogen. The hydrolysis renders trypsin active, which also has the ability to activate trypsinogen by cleaving Arg and Lys residues and then generating more trypsin [ 5 ]. In addition, trypsin also triggers a cascade activation of other proteolytic enzymes, and the resulting mixture of enzymes includes endopeptidases (trypsin, chymotrypsin, and elastase) and exopeptidases (carboxypeptidases A and B). Thus, the formation of trypsin by enteropeptidase is the essential activation step [ 2 ].
In this experiment, we describe a methodology adapted to the use of trypsinogen, chymotrypsinogen, and enteropeptidase from broilers, pigs, fishes, and ruminants due to the facility of obtaining these animals. Alternatively, other animals can be used such as guinea pigs, mice, or rabbits.
Generally, this experiment uses some hazardous reagents, like acetic acid, p-nitroanilide, N,N-dimethylformamide, and dimethylsulfoxide.
These reagents should be manipulated according to their handling instructions and disposed in accordance with local safety instructions.
6: Enzyme Activity and Protein Regulation - Biology
From humans to single-celled bacteria, the life of all organisms depends on the metabolic reactions occuring in their body. Proper functioning of an organism thus depends fundamentally on proper regulation of these biochemical reactions. And it is here that enzymes play a central role. Enzymes are proteins that catalyze (i.e., increaseor decrease the rates of)chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and they are converted into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at significant rates. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.
Like all catalysts, enzymes work by lowering the activation energy (Ea&Dagger) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze.
Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g., pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics.
Structures and Mechanisms
Ribbon diagram showing human carbonic anhydrase II. The grey sphere is the zinc cofactor in the active site. Diagram drawn from PDB 1MOO. Enzymes are generally globular proteins and range from just 62 amino acid residues in size to over 2,500 residues. A small number of RNA-based biological catalysts exist, with the most common being the ribosome these are referred to as either RNA-enzymes or ribozymes. The activities of enzymes are determined by their three-dimensional structure. However, although structure does determine function, predicting a novel enzyme's activity just from its structure is a very difficult problem that has not yet been solved.
Most enzymes are much larger than the substrates they act on, and only a small portion of the enzyme (around 3&ndash4 amino acids) is directly involved in catalysis. The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. Enzymes can also contain sites that bind cofactors, which are needed for catalysis. Some enzymes also have binding sites for small molecules, which are often direct or indirect products or substrates of the reaction catalyzed. This binding can serve to increase or decrease the enzyme's activity, providing a means for feedback regulation.
Like all proteins, enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties. Individual protein chains may sometimes group together to form a protein complex. Most enzymes can be denatured&mdashthat is, unfolded and inactivated&mdashby heating or chemical denaturants, which disrupt the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible.
Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible for this specificity.
Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome. These enzymes have "proof-reading" mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase, aminoacyl tRNA synthetases and ribosomes.
"Lock and Key" Model
Enzymes are very specific, and it was suggested by the Nobel laureate organic chemist Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve.
Diagrams to show the induced fit hypothesis of enzyme action.
In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined.
Enzymes can act in several ways, all of which lower &DeltaG+:
Lowering the activation energy by creating an environment in which the transition state is stabilized (e.g. straining the shape of a substrate&mdashby binding the transition-state conformation of the substrate/product molecules, the enzyme distorts the bound substrate(s) into their transition state form, thereby reducing the amount of energy required to complete the transition).
Lowering the energy of the transition state, but without distorting the substrate, by creating an environment with the opposite charge distribution to that of the transition state.
Providing an alternative pathway. For example, temporarily reacting with the substrate to form an intermediate ES complex, which would be impossible in the absence of the enzyme.
Reducing the reaction entropy change by bringing substrates together in the correct orientation to react. Considering &DeltaH&Dagger alone overlooks this effect.
Dynamics and Function
The internal dynamics of enzymes is linked to their mechanism of catalysis. Internal dynamics are the movement of parts of the enzyme's structure, such as individual amino acid residues, a group of amino acids, or even an entire protein domain. These movements occur at various time-scales ranging from femtoseconds to seconds. Networks of protein residues throughout an enzyme's structure can contribute to catalysis through dynamic motions. However, although these movements are important in binding and releasing substrates and products, it is not clear if protein movements help to accelerate the chemical steps in enzymatic reactions. These new insights also have implications in understanding allosteric effects and developing new drugs.
Allosteric transition of an enzyme between R and T states, stabilized by an agonist, an inhibitor and a substrate (the MWC model)
Allosteric sites are sites on the enzyme that bind to molecules in the cellular environment. The sites form weak, noncovalent bonds with these molecules, causing a change in the conformation of the enzyme. This change in conformation translates to the active site, which then affects the reaction rate of the enzyme. Allosteric interactions can both inhibit and activate enzymes and are a common way that enzymes are controlled in the body.
Cofactors and Coenzymes
Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules called cofactors to be bound for activity. Cofactors can be either inorganic (e.g., metal ions and iron-sulfur clusters) or organic compounds. Organic cofactors can be either prosthetic groups, which are tightly bound to an enzyme, or coenzymes, which are released from the enzyme's active site during the reaction. Coenzymes include NADH, NADPH and adenosine triphosphate. These molecules transfer chemical groups between enzymes.
Space-filling model of the coenzyme NADH
Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Tightly bound coenzymes can be called allosteric groups. Coenzymes transport chemical groups from one enzyme to another. The chemical groups carried include the hydride ion (H-) carried by NAD or NADP+, the phosphate group carried by adenosine triphosphate, the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosylmethionine.
Since coenzymes are chemically changed as a consequence of enzyme action, it is useful to consider coenzymes to be a special class of substrates, or second substrates, which are common to many different enzymes. For example, about 700 enzymes are known to use the coenzyme NADH.
Coenzymes are usually continuously regenerated and their concentrations maintained at a steady level inside the cell: for example, NADPH is regenerated through the pentose phosphate pathway. This continuous regeneration means that even small amounts of coenzymes are used very intensively. For example, the human body turns over its own weight in ATP each day.
Competitive inhibitors bind reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor. Substrate and inhibitor compete for the enzyme.
The coenzyme folic acid (left) and the anti-cancer drug methotrexate (right) are very similar in structure. As a result, methotrexate is a competitive inhibitor of many enzymes that use folates.
In competitive inhibition, the inhibitor and substrate compete for the enzyme (i.e., they can not bind at the same time). Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of folic acid and this drug are shown in the figure.
In uncompetitive inhibition the inhibitor can not bind to the free enzyme, but only to the ES-complex. The EIS-complex thus formed is enzymatically inactive. This type of inhibition is rare, but may occur in multimeric enzymes.
Non-competitive inhibitors can bind to the enzyme at the binding site at the same time as the substrate,but not to the active site. Both the EI and EIS complexes are enzymatically inactive. Because the inhibitor can not be driven from the enzyme by higher substrate concentration (in contrast to competitive inhibition), the apparent Vmax changes. But because the substrate can still bind to the enzyme, the Km stays the same.
This type of inhibition resembles the non-competitive, except that the EIS-complex has residual enzymatic activity. This type of inhibitor does not follow Michaelis-Menten equation.
In many organisms inhibitors may act as part of a feedback mechanism. If an enzyme produces too much of one substance in the organism, that substance may act as an inhibitor for the enzyme at the beginning of the pathway that produces it, causing production of the substance to slow down or stop when there is sufficient amount. This is a form of negative feedback. Enzymes which are subject to this form of regulation are often multimeric and have allosteric binding sites for regulatory substances. Their substrate/velocity plots are not hyperbolar, but sigmoidal (S-shaped).
Irreversible inhibitors react with the enzyme and form a covalent adduct with the protein. The inactivation is irreversible. These compounds include eflornithine a drug used to treat the parasitic disease sleeping sickness. Penicillin and Aspirin also act in this manner. With these drugs, the compound is bound in the active site and the enzyme then converts the inhibitor into an activated form that reacts irreversibly with one or more amino acid residues.
Enzymes serve a wide variety of functions inside living organisms. They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases. They also generate movement, with myosin hydrolysing ATP to generate muscle contraction and also moving cargo around the cell as part of the cytoskeleton. Other ATPases in the cell membrane are ion pumps involved in active transport. Enzymes are also involved in more exotic functions, such as luciferase generating light in fireflies. Viruses can also contain enzymes for infecting cells, such as the HIV integrase and reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.
An important function of enzymes is in the digestive systems of animals. Enzymes such as amylases and proteases break down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyse the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase to break down the cellulose cell walls of plant fiber.
Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel, this can allow more complex regulation: with for example a low constant activity being provided by one enzyme but an inducible high activity from a second enzyme.
Glycolytic enzymes and their functions in the metabolic pathway of glycolysis
Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps, nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. Consequently, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.
Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. Consequently, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been designed "from scratch" to catalyze reactions that do not occur in nature.
1. Comprehensive Laboratory Manual In Biology-XII 2. Biology Text For Class XII &ndash NCERT
Inhibitors: Molecules that bind to enzymes that prevent them from catalyzing reactions
- If binding involves covalent bonds, then inhibition is often irreversible
- If binding is weak, inhibition may be reversible
Competitive exclusion: The inhibitor binds to the same site as the substrate would, and blocks the substrate from binding to the enzyme
Non-competitive exclusion: The inhibitor binds somewhere other than the active site, changing the shape of the enzyme and blocking the substrate from binding