2BCK , 4B18 , 4MNQ
111-431: 7015 21752 ENSG00000164362 ENSMUSG00000021611 O14746 O70372 NM_001193376 NM_198253 NM_198254 NM_198255 NM_009354 NM_001362387 NM_001362388 NP_001180305 NP_937983 NP_033380 NP_001349316 NP_001349317 Telomerase reverse transcriptase (abbreviated to TERT , or hTERT in humans) is a catalytic subunit of the enzyme telomerase , which, together with
222-487: A catalytic triad , stabilize charge build-up on the transition states using an oxyanion hole , complete hydrolysis using an oriented water substrate. Enzymes are not rigid, static structures; instead they have complex internal dynamic motions – that is, movements of parts of the enzyme's structure such as individual amino acid residues, groups of residues forming a protein loop or unit of secondary structure , or even an entire protein domain . These motions give rise to
333-489: A conformational ensemble of slightly different structures that interconvert with one another at equilibrium . Different states within this ensemble may be associated with different aspects of an enzyme's function. For example, different conformations of the enzyme dihydrofolate reductase are associated with the substrate binding, catalysis, cofactor release, and product release steps of the catalytic cycle, consistent with catalytic resonance theory . Substrate presentation
444-466: A cytotoxic T-cell reaction against telomerase-positive tumor cells in vitro . The response is mediated by dendritic cells , which can display hTERT-associated antigens on MHC class I and II receptors following adenoviral transduction of an hTERT plasmid into dendritic cells, which mediate T-cell responses. Dendritic cells are then able to present telomerase-associated antigens even with undetectable amounts of telomerase activity, as long as
555-468: A 24% increase in lifespan, without any increase in cancer. Paradoxically, genetic variants in the TERT locus, which are associated with longer leukocyte telomere length, are associated with faster epigenetic aging rates in blood according to a molecular biomarker of aging known as epigenetic clock . Similarly, human TERT expression did not arrest epigenetic aging in human fibroblasts. The hTERT gene has become
666-526: A balance within the cell such that the telomeres are of sufficient length for their function and yet, at the same time, not contribute to aberrant telomere elongation. High expression of hTERT is also often used as a landmark for pluripotency and multipotency state of embryonic and adult stem cells. Over-expression of hTERT was found to immortalize certain cell types as well as impart different interesting properties to different stem cells. hTERT immortalizes various normal cells in culture, thereby endowing
777-474: 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 . Conversely, some enzymes display enzyme promiscuity , having broad specificity and acting on
888-435: A low density of tri-methylated histones H3K9 and H4K20 at telomeres, as well as an increased detectable amount of TERT transcripts and protein activity. Without the restoration of TERT and associated telomerase proteins, the efficiency of iPS cells would be drastically reduced. iPS cells would also lose the ability to self-renew and would eventually senesce . DKC ( dyskeratosis congenita ) patients are all characterized by
999-451: A main focus for gene therapy involving cancer due to its expression in tumor cells but not somatic adult cells. One method is to prevent the translation of hTERT mRNA through the introduction of siRNA , which are complementary sequences that bind to the mRNA preventing processing of the gene post transcription . This method does not eliminate telomerase activity, but it does lower telomerase activity and levels of hTERT mRNA seen in
1110-523: A mechanism for increased cancer incidence in obese individuals. There are several other regulatory mechanisms that are altered or aberrant in cancer cells, including the Ras signaling pathway and other transcriptional regulators. Phosphorylation is also a key process of post-transcriptional modification that regulates mRNA expression and cellular localization. Clearly, there are many regulatory mechanisms of activation and repression of hTERT and telomerase activity in
1221-449: A pivotal role in the (MLP) of subgroup C human adenoviruses, in species with a deficient CAAT sequence. The transcription initiation at mutant MLP species was significantly reduced compared with that of the wild type or species in which there was a CAAT mutant. The failure to restore the normally functional adenoviruses, exhibited by a CAAT box, is consistent with the idea that the CAAT box plays
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#17328756027001332-692: A prodrug only activated by the viral enzyme, specific targeting of cells expressing hTERT can be achieved. By using the hTERT promoter, only cells expressing hTERT will be affected and this allows for specific targeting of tumor cells. Aside from cancer therapies, the hTERT gene has been used to promote the growth of hair follicles. A schematic animation for gene therapy is shown as follows. Telomerase reverse transcriptase has been shown to interact with: Enzyme Enzymes ( / ˈ ɛ n z aɪ m z / ) are proteins that act as biological catalysts by accelerating chemical reactions . The molecules upon which enzymes may act are called substrates , and
1443-464: A quantitative theory of enzyme kinetics, which is referred to as Michaelis–Menten kinetics . The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis–Menten complex in their honor. The enzyme then catalyzes the chemical step in
1554-439: A range of different physiologically relevant substrates. Many enzymes possess small side activities which arose fortuitously (i.e. neutrally ), which may be the starting point for the evolutionary selection of a new function. To explain the observed specificity of enzymes, in 1894 Emil Fischer proposed that both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This
1665-650: A role in inhibiting the mTOR pathway via down-regulation of phosphorylation. The mTOR pathway is very important in regulating protein synthesis and it interacts with telomerase to increase its expression. Several other chemicals have been found to inhibit telomerase activity and are currently being tested as potential clinical treatment options such as nucleoside analogues , retinoic acid derivatives, quinolone antibiotics , and catechin derivatives . There are also other molecular genetic-based methods of inhibiting telomerase, such as antisense therapy and RNA interference . hTERT peptide fragments have been shown to induce
1776-555: A small portion of their capabilities, since the number of theoretically combinations of NF-Y complexes is so large and only a small portion can actually be created (less than 10% of all possible interactions were confirmed in both directions in yeast). Another aspect of the CCAAT binding motif is the CCAAT/enhancer binding proteins (C/EBPs). They are a group of transcription factors of 6 members (α-ζ), which are highly conserved and bind to
1887-451: A species' normal level; as a result, enzymes from bacteria living in volcanic environments such as hot springs are prized by industrial users for their ability to function at high temperatures, allowing enzyme-catalysed reactions to be operated at a very high rate. Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase , to over 2,500 residues in
1998-446: A steady level inside the cell. For example, NADPH is regenerated through the pentose phosphate pathway and S -adenosylmethionine by methionine adenosyltransferase . This continuous regeneration means that small amounts of coenzymes can be used very intensively. For example, the human body turns over its own weight in ATP each day. As with all catalysts, enzymes do not alter the position of
2109-496: A strong correlation between telomerase activity and malignant tumors or cancerous cell lines. Not all types of human cancer have increased telomerase activity. 90% of cancers are characterized by increased telomerase activity. Lung cancer is the most well characterized type of cancer associated with telomerase. There is a lack of substantial telomerase activity in some cell types such as primary human fibroblasts , which become senescent after about 30–50 population doublings. There
2220-648: A suitable method for treating circulatory ailments such as bone marrow degeneration and low blood count linked with DKC and other telomerase-deficient conditions. As organisms age and cells proliferate, telomeres shorten with each round of replication. Cells restricted to a specific lineage are capable of division only a set number of times, set by the length of telomeres, before they senesce . Depletion and uncapping of telomeres has been linked to organ degeneration, failure , and fibrosis due to progenitors' becoming quiescent and unable to differentiate . Using an in vivo TERT deficient mouse model , reactivation of
2331-442: A thermodynamically unfavourable one so that the combined energy of the products is lower than the substrates. For example, the hydrolysis of ATP is often used to drive other chemical reactions. Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are commonly obtained from enzyme assays . In 1913 Leonor Michaelis and Maud Leonora Menten proposed
SECTION 20
#17328756027002442-404: A toxin, an apoptotic factor, or a viral protein. Toxins such as diphtheria toxin interfere with cellular processes and eventually induce apoptosis. Apoptotic death factors like FADD (Fas-Associated protein with Death Domain) can be used to force cells expressing hTERT to undergo apoptosis. Viral proteins like viral thymidine kinase can be used for specific targeting of a drug. By introducing
2553-458: A vital role in the adenovirus MLP and is preferred over other transcriptional elements. These core binding factors, or nuclear factors (NF-Y), are composed of three subunits – NF-YA, NF-YB, and NF-YC. Whereas in animals each NF-Y subunit is encoded by a single gene, there has been a diversification in plants in both structure and function. Families of NF-Y consist of between eight and 39 members per subunit. A large reason for this diversification
2664-457: Is k cat , also called the turnover number , which is the number of substrate molecules handled by one active site per second. The efficiency of an enzyme can be expressed in terms of k cat / K m . This is also called the specificity constant and incorporates the rate constants for all steps in the reaction up to and including the first irreversible step. Because the specificity constant reflects both affinity and catalytic ability, it
2775-838: Is orotidine 5'-phosphate decarboxylase , which allows a reaction that would otherwise take millions of years to occur in milliseconds. Chemically, enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity, and activators are molecules that increase activity. Many therapeutic drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly outside its optimal temperature and pH , and many enzymes are (permanently) denatured when exposed to excessive heat, losing their structure and catalytic properties. Some enzymes are used commercially, for example, in
2886-745: Is a ribonucleoprotein polymerase that maintains telomere ends by addition of the telomere repeat TTAGGG. The enzyme consists of a protein component with reverse transcriptase activity, encoded by this gene, and an RNA component that serves as a template for the telomere repeat. Telomerase expression plays a role in cellular senescence, as it is normally repressed in postnatal somatic cells , resulting in progressive shortening of telomeres. Studies in mice suggest that telomerase also participates in chromosomal repair, since de novo synthesis of telomere repeats may occur at double-stranded breaks . Alternatively spliced variants encoding different isoforms of telomerase reverse transcriptase have been identified;
2997-543: Is a feature frequently found before eukaryote coding regions, but is not found in prokaryotes. In the direction of transcription of the template strand, the consensus sequence , or the calculated order of the most frequent residues, for the CAAT box was 3'-TG ATTGG (T/C)(T/C)(A/G)-5'. The use of parentheses denotes that either base is present, but it is not specified as to their relative frequencies. For example, "(T/C)" would mean that either thymine or cytosine are preferentially selected for. Within metazoa (animal kingdom),
3108-732: Is a major limiting component of the telomerase complex and a deficiency of intact hTERT impedes the activity of telomerase, making iPS cells an unsuitable pathway towards therapy for telomere-deficient disorders. Although the mechanism is not fully understood, exposure of TERT-deficient hematopoietic cells to androgens resulted in an increased level of TERT activity. Cells with a heterozygous TERT mutation, like those in DKC (dyskeratosis congenita) patients, which normally exhibit low baseline levels of TERT, could be restored to normal levels comparable to control cells. TERT mRNA levels are also increased with exposure to androgens. Androgen therapy may become
3219-421: Is a process where the enzyme is sequestered away from its substrate. Enzymes can be sequestered to the plasma membrane away from a substrate in the nucleus or cytosol. Or within the membrane, an enzyme can be sequestered into lipid rafts away from its substrate in the disordered region. When the enzyme is released it mixes with its substrate. Alternatively, the enzyme can be sequestered near its substrate to activate
3330-401: Is a susceptibility gene for development of many cancers, including lung cancer. Telomerase activity is associated with the number of times a cell can divide playing an important role in the immortality of cell lines, such as cancer cells . The enzyme complex acts through the addition of telomeric repeats to the ends of chromosomal DNA. This generates immortal cancer cells. In fact, there is
3441-593: Is adjacent to the A1 domain by a conserved linker sequence is a sequence of 21 amino acids vital in the specific DNA to CCAAT box binding. The A1 and A2 domains are conserved towards the C-terminus of mammals, but occupy a more central region in plant NF-YA subunits. In plants, the NF-YA subunit has evolved to regulate the development of a facultative root organ only present in leguminous plants and shown to be expressed in root tissue. It
Telomerase reverse transcriptase - Misplaced Pages Continue
3552-522: Is also evidence that telomerase activity is increased in tissues, such as germ cell lines, that are self-renewing. Normal somatic cells , on the other hand, do not have detectable telomerase activity. Since the catalytic component of telomerase is its reverse transcriptase, hTERT, and the RNA component hTERC, hTERT is an important gene to investigate in terms of cancer and tumorigenesis . The hTERT gene has been examined for mutations and their association with
3663-980: Is because of gene duplications and tandem duplications, which have helped contribute to the larger family sizes of NF-Y compared to the single encoded animal nuclear factors. Each subunit contains an evolutionarily conserved part – the C-terminal of NF-YA, the central part of NF-YB, and the N-terminal of NF-YC, greater than 70% of these across species remains conserved. Neighboring regions however are generally not conserved. The NF-YA family encodes transcription factors that are variable in length (between 207 and 347 amino acids for M. truncatula ). The NF-YA proteins are generally characterized by two domains that are strongly conserved in all higher eukaryotes investigated to date. The first domain (A1) contains 20 amino acids that forms an alpha helix that appears significant in its interactions with NF-YB and NF-YC. The second domain (A2)
3774-437: Is described by "EC" followed by a sequence of four numbers which represent the hierarchy of enzymatic activity (from very general to very specific). That is, the first number broadly classifies the enzyme based on its mechanism while the other digits add more and more specificity. The top-level classification is: These sections are subdivided by other features such as the substrate, products, and chemical mechanism . An enzyme
3885-749: Is fully specified by four numerical designations. For example, hexokinase (EC 2.7.1.1) is a transferase (EC 2) that adds a phosphate group (EC 2.7) to a hexose sugar, a molecule containing an alcohol group (EC 2.7.1). Sequence similarity . EC categories do not reflect sequence similarity. For instance, two ligases of the same EC number that catalyze exactly the same reaction can have completely different sequences. Independent of their function, enzymes, like any other proteins, have been classified by their sequence similarity into numerous families. These families have been documented in dozens of different protein and protein family databases such as Pfam . Non-homologous isofunctional enzymes . Unrelated enzymes that have
3996-414: Is higher than what actually happens since some subunits have specific binding patterns. Functional analyses on NF-Y encoding genes in plants have shown, as a result of their evolutionary diversification relative to their animal counterparts, have acquired diverse specific functions, such as embryo development, flowering time control, ER-stress, drought stress, and nodule and root development. This may only be
4107-423: Is often associated with cancers and tumor formation. The regulation of hTERT is extremely important to the maintenance of stem and cancer cells and can be used in multiple ways in the field of regenerative medicine . hTERT is often up-regulated in cells that divide rapidly, including both embryonic stem cells and adult stem cells . It elongates the telomeres of stem cells , which, as a consequence, increases
4218-473: Is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase . Examples are lactase , alcohol dehydrogenase and DNA polymerase . Different enzymes that catalyze the same chemical reaction are called isozymes . The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers (for "Enzyme Commission") . Each enzyme
4329-418: Is often referred to as "the lock and key" model. This early model explains enzyme specificity, but fails to explain the stabilization of the transition state that enzymes achieve. In 1958, Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with
4440-462: Is only one of several important kinetic parameters. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis–Menten constant ( K m ), which is the substrate concentration required for an enzyme to reach one-half its maximum reaction rate; generally, each enzyme has a characteristic K M for a given substrate. Another useful constant
4551-404: Is seen. This is shown in the saturation curve on the right. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES complex. At the maximum reaction rate ( V max ) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme. V max
Telomerase reverse transcriptase - Misplaced Pages Continue
4662-403: Is the ribosome which is a complex of protein and catalytic RNA components. Enzymes must bind their substrates before they can catalyse any chemical reaction. Enzymes are usually very specific as to what substrates they bind and then the chemical reaction catalysed. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic / hydrophobic characteristics to
4773-453: Is through demethylation of histones proximal to the promoter region, imitating the low density of trimethylated histones seen in embryonic stem cells . This allows for the recruitment of histone acetyltransferase (HAT) to unwind the sequence allowing for transcription of the gene. Telomere deficiency is often linked to aging, cancers and the conditions dyskeratosis congenita (DKC) and Cri du chat . Meanwhile, over-expression of hTERT
4884-790: Is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 10 to 10 (M s ). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect . Example of such enzymes are triose-phosphate isomerase , carbonic anhydrase , acetylcholinesterase , catalase , fumarase , β-lactamase , and superoxide dismutase . The turnover of such enzymes can reach several million reactions per second. But most enzymes are far from perfect:
4995-611: The DNA polymerases ; here the holoenzyme is the complete complex containing all the subunits needed for activity. Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Coenzymes transport chemical groups from one enzyme to another. Examples include NADH , NADPH and adenosine triphosphate (ATP). Some coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP), and tetrahydrofolate (THF), are derived from vitamins . These coenzymes cannot be synthesized by
5106-460: The GC box is known for binding general transcription factors. Both of these consensus sequences belong to the regulatory promoter . Full gene expression occurs when transcription activator proteins bind to each module within the regulatory promoter. Protein specific binding is required for the CCAAT box activation. These proteins are known as CCAAT box binding proteins/CCAAT box binding factors. A CCAAT box
5217-458: The cytoplasm . Higher success rates were seen in vitro when combining the use of antisense hTERT sequences with the introduction of a tumor-suppressing plasmid by adenovirus infection such as PTEN . Another method that has been studied is manipulating the hTERT promoter to induce apoptosis in tumor cells. Plasmid DNA sequences can be manufactured using the hTERT promoter followed by genes encoding for specific proteins. The protein can be
5328-511: The law of mass action , which is derived from the assumptions of free diffusion and thermodynamically driven random collision. Many biochemical or cellular processes deviate significantly from these conditions, because of macromolecular crowding and constrained molecular movement. More recent, complex extensions of the model attempt to correct for these effects. Enzyme reaction rates can be decreased by various types of enzyme inhibitors. A competitive inhibitor and substrate cannot bind to
5439-451: The telomerase RNA component (TERC), comprises the most important unit of the telomerase complex. Telomerases are part of a distinct subgroup of RNA-dependent polymerases. Telomerase lengthens telomeres in DNA strands, thereby allowing senescent cells that would otherwise become postmitotic and undergo apoptosis to exceed the Hayflick limit and become potentially immortal, as is often
5550-514: The ATTGG motif with two adenine residues (AA) on its 5' end and G(A/G) on its 3' end, seems to have inhibited formation of the CBF-DNA complex and subsequently occurred in only 1% of the promoter sequences. In another experiment performed with the major late promoter (MLP) of adenoviruses from a variety of host species, it was shown that the mutation of the CAAT box and CCAAT sequence, which is thought to play
5661-436: The CAAT box, is located about 60–100 bases upstream (towards the 5' end), however no less than 27 base pairs away, from the initial transcription site or a eukaryote gene in which a complex of general transcription factors bind with RNA polymerase II prior to the initiation of transcription. It is essential to the transcription that these core binding factors (also referred to as nuclear factor Y or NF-Y) are able to bind to
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#17328756027005772-417: The CCAAT motif. Experiments in many laboratories have shown that mutations to the CCAAT motif that cause a loss of CBF binding also decreases transcriptional activity in these promoters, suggesting that CBF-CCAAT complexes are essential for optimum transcriptional activity. In an experiment done with core binding factors (CBF) and DNA complexes, researchers were able to determine the preferential sequences of
5883-475: The CCAAT motif. While research on these binding proteins is relatively recent, their function has been shown to have vital roles in cellular proliferation and differentiation, metabolism , inflammation, and immunity in various cells, but specifically hepatocytes , adipocytes , and hematopoietic cells . For example, in adipocytes, this has been shown in a variety of experiments with mice: ectopic expression of these C/EBPs (C/EBPα and C/EBPβ) were able to initiate
5994-981: The HFM that is prevalent in NF-YB proteins. It has also been shown to be involved in flowering time in certain plants (overexpression leads to earlier flowering) where its influence is potentially regulated by the binding of the protein CONSTANS (CO) to the NF-YC subunit. Because of the evolutionary change in NF-Y encoding genes in plants, they subsequently have a large range of potential trimeric complexes. For example, in Arabidopsis , 36 NF-Y transcription factor subunits (including 10 NF-YA, 13 NF-YB, and 13 NF-YC subunits) have been identified and which could theoretically form 1690 unique complexes (which contains one of each type of subunit). This number, of course
6105-553: The NF-YA subunit (90–240 amino acids in "M. truncatula"). They have been characterized with a structure and amino acid composition similar to the histone fold motif (HFM). This is composed of three alpha-helices separated by two beta strand-loop domains. Similar to NF-YA, NF-YB has been shown to also improve drought resistance when overexpressed and also the promotion of flowering in Arabidopsis . The NF-YC proteins are an intermediate size between that of NF-YA and NF-YB proteins (117–292 amino acids in M. truncatula ) and also contain
6216-442: The TERT gene in quiescent populations in multiple organs reactivated telomerase and restored the cells’ abilities to differentiate . Reactivation of TERT down-regulates DNA damage signals associated with cellular mitotic checkpoints allowing for proliferation and elimination of a degenerative phenotype . In another study, introducing the TERT gene into healthy one-year-old mice using an engineered adeno-associated virus led to
6327-400: The ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties. Enzymes are known to catalyze more than 5,000 biochemical reaction types. Other biocatalysts are catalytic RNA molecules , also called ribozymes . They are sometimes described as a type of enzyme rather than being like an enzyme, but even in
6438-417: The ability to self-renew indefinitely and contribute to all three germ layers when implanted into a blastocyst or use in teratoma formation. Early development of iPS cell lines were not efficient, as they yielded up to 5% of somatic cells successfully reprogrammed into a stem cell-like state. By using immortalized somatic cells ( differentiated cells with hTERT upregulated ), iPS cell reprogramming
6549-437: The active site and are involved in catalysis. For example, flavin and heme cofactors are often involved in redox reactions. Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins . An enzyme together with the cofactor(s) required for activity is called a holoenzyme (or haloenzyme). The term holoenzyme can also be applied to enzymes that contain multiple protein subunits, such as
6660-502: The active site. Organic cofactors can be either coenzymes , which are released from the enzyme's active site during the reaction, or prosthetic groups , which are tightly bound to an enzyme. Organic prosthetic groups can be covalently bound (e.g., biotin in enzymes such as pyruvate carboxylase ). An example of an enzyme that contains a cofactor is carbonic anhydrase , which uses a zinc cofactor bound as part of its active site. These tightly bound ions or molecules are usually found in
6771-407: The animal fatty acid synthase . Only a small portion of their structure (around 2–4 amino acids) is directly involved in catalysis: the catalytic site. This catalytic site is located next to one or more binding sites where residues orient the substrates. The catalytic site and binding site together compose the enzyme's active site . The remaining majority of the enzyme structure serves to maintain
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#17328756027006882-578: The average values of k c a t / K m {\displaystyle k_{\rm {cat}}/K_{\rm {m}}} and k c a t {\displaystyle k_{\rm {cat}}} are about 10 5 s − 1 M − 1 {\displaystyle 10^{5}{\rm {s}}^{-1}{\rm {M}}^{-1}} and 10 s − 1 {\displaystyle 10{\rm {s}}^{-1}} , respectively. Michaelis–Menten kinetics relies on
6993-460: The binding site for the RNA transcription factor , and is typically accompanied by a conserved consensus sequence. It is an invariant DNA sequence at about minus 70 base pairs from the origin of transcription in many eukaryotic promoters . Genes that have this element seem to require it for the gene to be transcribed in sufficient quantities. It is frequently absent from genes that encode proteins used in virtually all cells. This box along with
7104-502: The body de novo and closely related compounds (vitamins) must be acquired from the diet. The chemical groups carried include: 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 1000 enzymes are known to use the coenzyme NADH. Coenzymes are usually continuously regenerated and their concentrations maintained at
7215-437: The case with cancerous cells. To be specific, TERT is responsible for catalyzing the addition of nucleotides in a TTAGGG sequence to the ends of a chromosome 's telomeres . This addition of repetitive DNA sequences prevents degradation of the chromosomal ends following multiple rounds of replication. hTERT absence (usually as a result of a chromosomal mutation ) is associated with the disorder Cri du chat . Telomerase
7326-537: The cell, providing methods of immortalization in cancer cells. If increased telomerase activity is associated with malignancy , then possible cancer treatments could involve inhibiting its catalytic component, hTERT, to reduce the enzyme's activity and cause cell death. Since normal somatic cells do not express TERT, telomerase inhibition in cancer cells can cause senescence and apoptosis without affecting normal human cells. It has been found that dominant-negative mutants of hTERT could reduce telomerase activity within
7437-415: The cell. This led to apoptosis and cell death in cells with short telomere lengths, a promising result for cancer treatment. Although cells with long telomeres did not experience apoptosis, they developed mortal characteristics and underwent telomere shortening. Telomerase activity has also been found to be inhibited by phytochemicals such as isoprenoids , genistein , curcumin , etc. These chemicals play
7548-471: The chemical equilibrium of the reaction. In the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly. For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants: The rate of a reaction is dependent on the activation energy needed to form the transition state which then decays into products. Enzymes increase reaction rates by lowering
7659-425: The conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by which these occurred had not been identified. French chemist Anselme Payen was the first to discover an enzyme, diastase , in 1833. A few decades later, when studying the fermentation of sugar to alcohol by yeast , Louis Pasteur concluded that this fermentation was caused by a vital force contained within
7770-408: The core binding factor (CBF)-DNA complex retains a high degree of conservation within the CCAAT binding motif, as well as the sequences flanking this pentameric motif. The CCAAT motif in plants (spinach was used in an experiment) differs slightly from metazoa in that it is actually a CAAT binding motif; the promoter lacks one of the two C residues from the pentameric motif, and the artificial addition of
7881-444: The decades since ribozymes' discovery in 1980–1982, the word enzyme alone often means the protein type specifically (as is used in this article). An enzyme's specificity comes from its unique three-dimensional structure . Like all catalysts, enzymes increase the reaction rate by lowering its activation energy . Some enzymes can make their conversion of substrate to product occur many millions of times faster. An extreme example
7992-452: The defective maintenance of telomeres leading to problems with stem cell regeneration. iPS cells derived from DKC patients with a heterozygous mutation on the TERT gene display a 50% reduction in telomerase activity compared to wild type iPS cells. Conversely, mutations on the TERC gene (RNA portion of telomerase complex) can be overcome by up-regulation due to reprogramming as long as
8103-568: The different types of stem cells. Hence, not all stem cells will have increased stem-cell properties. For example, research has shown that telomerase can be upregulated in CD34+ Umbilical Cord Blood Cells through hTERT over-expression. The survival of these stem cells was enhanced, although there was no increase in the amount of population doubling. Deregulation of telomerase expression in somatic cells may be involved in oncogenesis . Genome-wide association studies suggest TERT
8214-408: The differentiation capacity of adult stem cells may be dependent on telomerase activities. Therefore, over-expression of hTERT, which is akin to increasing telomerase activities, may create adult stem cells with a larger capacity for differentiation and hence, a larger capacity for treatment. Increasing the telomerase activities in stem cells gives different effects depending on the intrinsic nature of
8325-483: The differentiation programs of the cell, even in the absence of adipogenic hormones , or the differentiation of preadipocytes to adipocytes (or fat cells). In addition, an overabundance of these C/EBPs (specifically, C/EBPδ) causes an accelerated response. And furthermore, in cells lacking C/EBP or in C/EBP-deficient mice, both are unable to undergo adipogenesis. This results in the mice dying from hypoglycemia , or
8436-433: The energy of the transition state. First, binding forms a low energy enzyme-substrate complex (ES). Second, the enzyme stabilises the transition state such that it requires less energy to achieve compared to the uncatalyzed reaction (ES ). Finally the enzyme-product complex (EP) dissociates to release the products. Enzymes can couple two or more reactions, so that a thermodynamically favorable reaction can be used to "drive"
8547-587: The enzyme urease was a pure protein and crystallized it; he did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively demonstrated by John Howard Northrop and Wendell Meredith Stanley , who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin . These three scientists were awarded the 1946 Nobel Prize in Chemistry. The discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography . This
8658-483: The enzyme at the same time. Often competitive inhibitors strongly resemble the real substrate of the enzyme. For example, the drug methotrexate is a competitive inhibitor of the enzyme dihydrofolate reductase , which catalyzes the reduction of dihydrofolate to tetrahydrofolate. The similarity between the structures of dihydrofolate and this drug are shown in the accompanying figure. This type of inhibition can be overcome with high substrate concentration. In some cases,
8769-422: The enzyme converts the substrates into different molecules known as products . Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and the field of pseudoenzyme analysis recognizes that during evolution, some enzymes have lost
8880-403: The enzyme. As a result, the substrate does not simply bind to a rigid active site; the amino acid side-chains that make up the active site are molded into the precise positions that enable the enzyme to perform its catalytic function. 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
8991-427: The enzyme. For example, the enzyme can be soluble and upon activation bind to a lipid in the plasma membrane and then act upon molecules in the plasma membrane. Allosteric sites are pockets on the enzyme, distinct from the active site, that bind to molecules in the cellular environment. These molecules then cause a change in the conformation or dynamics of the enzyme that is transduced to the active site and thus affects
9102-461: The full-length sequence of some variants has not been determined. Alternative splicing at this locus is thought to be one mechanism of regulation of telomerase activity. The hTERT gene, located on chromosome 5, consists of 16 exons and 15 introns spanning 35 kb. The core promoter of hTERT includes 330 base pairs upstream of the translation start site (AUG since it is RNA by using the words "exons" and "introns"), as well as 37 base pairs of exon 2 of
9213-407: The hTERT gene is intact and functional. Lastly, iPS cells generated with DKC cells with a mutated dyskerin (DKC1) gene cannot assemble the hTERT/RNA complex and thus do not have functional telomerase. The functionality and efficiency of a reprogrammed iPS cell is determined by the ability of the cell to re-activate the telomerase complex and elongate its telomeres allowing for self-renewal. hTERT
9324-531: The hTERT gene. The hTERT promoter is GC-rich and lacks TATA and CAAT boxes but contains many sites for several transcription factors giving indication of a high level of regulation by multiple factors in many cellular contexts. Transcription factors that can activate hTERT include many oncogenes (cancer-causing genes) such as c-Myc , Sp1 , HIF-1 , AP2 , and many more, while many cancer suppressing genes such as p53 , WT1 , and Menin produce factors that suppress hTERT activity. Another form of up-regulation
9435-422: The hTERT plasmid is present. Immunotherapy against telomerase-positive tumor cells is a promising field in cancer research that has been shown to be effective in in vitro and mouse model studies. Induced pluripotent stem cells (iPS cells) are somatic cells that have been reprogrammed into a stem cell -like state by the introduction of four factors ( Oct3/4 , Sox2 , Klf4 , and c-Myc ). iPS cells have
9546-463: The hTERT promoter were first identified in melanoma and have subsequently been shown to be the most common noncoding mutations in cancer. Glycogen synthase kinase 3 ( GSK3 ) seems to be over-expressed in most cancer cells. GSK3 is involved in promoter activation through controlling a network of transcription factors . Leptin is also involved in increasing mRNA expression of hTERT via signal transducer and activation of transcription 3 ( STAT3 ), proposing
9657-446: The inhibitor can bind to a site other than the binding-site of the usual substrate and exert an allosteric effect to change the shape of the usual binding-site. CAAT box In molecular biology , a CCAAT box (also sometimes abbreviated a CAAT box or CAT box ) is a distinct pattern of nucleotides with GGCCAATCT consensus sequence that occur upstream by 60–100 bases to the initial transcription site. The CAAT box signals
9768-647: The length of the telomeres while maintaining stem cell-like abilities to differentiate. Reactivation of TERT activity can be achieved using only three of the four reprogramming factors described by Takahashi and Yamanaka: To be specific, Oct3/4 , Sox2 and Klf4 are essential, whereas c-Myc is not. However, this study was done with cells containing endogenous levels of c-Myc that may have been sufficient for reprogramming. Telomere length in healthy adult cells elongates and acquires epigenetic characteristics similar to those of ES cells when reprogrammed as iPS cells. Some epigenetic characteristics of ES cells include
9879-411: The lifespan of the stem cells by allowing for indefinite division without shortening of telomeres. Therefore, it is responsible for the self-renewal properties of stem cells. Telomerase are found specifically to target shorter telomere over longer telomere, due to various regulatory mechanisms inside the cells that reduce the affinity of telomerase to longer telomeres. This preferential affinity maintains
9990-468: The mixture. He named the enzyme that brought about the fermentation of sucrose " zymase ". In 1907, he received the Nobel Prize in Chemistry for "his discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named according to the reaction they carry out: the suffix -ase is combined with the name of the substrate (e.g., lactase is the enzyme that cleaves lactose ) or to
10101-528: The precise orientation and dynamics of the active site. In some enzymes, no amino acids are directly involved in catalysis; instead, the enzyme contains sites to bind and orient catalytic cofactors . Enzyme structures may also contain allosteric sites where the binding of a small molecule causes a conformational change that increases or decreases activity. A small number of RNA -based biological catalysts called ribozymes exist, which again can act alone or in complex with proteins. The most common of these
10212-403: The promoter in a region over and immediately adjacent to the CAAT box, and two regions on either side of the CAAT box. By using PCR -mediated random binding selection process, researchers were able to show that the sequence "3' - (T/C)G ATTGG (T/C)(T/C)(A/G) - 5'" immediately flanking the ATTGG region (CCAAT in the complementary strand) was preferentially selected on the coding strand (opposite of
10323-406: The reaction and releases the product. This work was further developed by G. E. Briggs and J. B. S. Haldane , who derived kinetic equations that are still widely used today. Enzyme rates depend on solution conditions and substrate concentration . To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation
10434-733: The reaction rate of the enzyme. In this way, allosteric interactions can either inhibit or activate enzymes. Allosteric interactions with metabolites upstream or downstream in an enzyme's metabolic pathway cause feedback regulation, altering the activity of the enzyme according to the flux through the rest of the pathway. Some enzymes do not need additional components to show full activity. 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 (e.g., flavin and heme ). These cofactors serve many purposes; for instance, metal ions can help in stabilizing nucleophilic species within
10545-444: The risk of contracting cancer. Over two hundred combinations of hTERT polymorphisms and cancer development have been found. There were several different types of cancer involved, and the strength of the correlation between the polymorphism and developing cancer varied from weak to strong. The regulation of hTERT has also been researched to determine possible mechanisms of telomerase activation in cancer cells. Importantly, mutations in
10656-410: The same enzymatic activity have been called non-homologous isofunctional enzymes . Horizontal gene transfer may spread these genes to unrelated species, especially bacteria where they can replace endogenous genes of the same function, leading to hon-homologous gene displacement. Enzymes are generally globular proteins , acting alone or in larger complexes . The sequence of the amino acids specifies
10767-488: The second C has no significant effects on binding activity. Some sequences lack the CAAT-box completely. Secondly, the surrounding nucleotides in plants do not match the consensus sequence above determined by Bi et al. The CAAT box is what is known as a core promoter, also known as the basal promoter or simply the promoter , is a region of DNA that initiates transcription of a particular gene. This region, in particular for
10878-851: The self-renewal properties of stem cells to non-stem cell cultures. There are multiple ways in which immortalization of non-stem cells can be achieved, one of which being via the introduction of hTERT into the cells. Differentiated cells often express hTERC and TP1, a telomerase-associated protein that helps form the telomerase assembly, but does not express hTERT. Hence, hTERT acts as the limiting factor for telomerase activity in differentiated cells. However, with hTERT over-expression, active telomerase can be formed in differentiated cells. This method has been used to immortalize prostate epithelial and stromal-derived cells, which are typically difficult to culture in vitro . hTERT introduction allows in vitro culture of these cells and available for possible future research. The introduction of hTERT has an advantage over
10989-412: The structure which in turn determines the catalytic activity of the enzyme. Although structure determines function, a novel enzymatic activity cannot yet be predicted from structure alone. Enzyme structures unfold ( denature ) when heated or exposed to chemical denaturants and this disruption to the structure typically causes a loss of activity. Enzyme denaturation is normally linked to temperatures above
11100-519: The substrate is completely bound, at which point the final shape and charge distribution is determined. Induced fit may enhance the fidelity of molecular recognition in the presence of competition and noise via the conformational proofreading mechanism. Enzymes can accelerate reactions in several ways, all of which lower the activation energy (ΔG , Gibbs free energy ) Enzymes may use several of these mechanisms simultaneously. For example, proteases such as trypsin perform covalent catalysis using
11211-405: The substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemoselective , regioselective and stereospecific . Some of the enzymes showing the highest specificity and accuracy are involved in the copying and expression of the genome . Some of these enzymes have " proof-reading " mechanisms. Here, an enzyme such as DNA polymerase catalyzes a reaction in
11322-399: The synthesis of antibiotics . Some household products use enzymes to speed up chemical reactions: enzymes in biological washing powders break down protein, starch or fat stains on clothes, and enzymes in meat tenderizer break down proteins into smaller molecules, making the meat easier to chew. By the late 17th and early 18th centuries, the digestion of meat by stomach secretions and
11433-414: The template strand). This was shown using an oligonucleotide sequence (R1) which contained 27 random nucleotides, flanked by a defined 20 nucleotide sequence on each side. While no single nucleotide was selected in every clone on either side of the ATTGG motif (CCAAT in the complementary strand), there were several nucleotides in positions selected with high frequency. Most notably from the sequence above
11544-438: The type of reaction (e.g., DNA polymerase forms DNA polymers). The biochemical identity of enzymes was still unknown in the early 1900s. Many scientists observed that enzymatic activity was associated with proteins, but others (such as Nobel laureate Richard Willstätter ) argued that proteins were merely carriers for the true enzymes and that proteins per se were incapable of catalysis. In 1926, James B. Sumner showed that
11655-627: The use of viral protein for immortalization in that it does not involve the inactivation of tumor suppressor gene , which might lead to cancer formation. Over-expression of hTERT in stem cells changes the properties of the cells. hTERT over-expression increases the stem cell properties of human mesenchymal stem cells . The expression profile of mesenchymal stem cells converges towards embryonic stem cells, suggesting that these cells may have embryonic stem cell-like properties. However, it has been observed that mesenchymal stem cells undergo decreased levels of spontaneous differentiation . This suggests that
11766-486: The yeast cells called "ferments", which were thought to function only within living organisms. He wrote that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells, not with the death or putrefaction of the cells." In 1877, German physiologist Wilhelm Kühne (1837–1900) first used the term enzyme , which comes from Ancient Greek ἔνζυμον (énzymon) ' leavened , in yeast', to describe this process. The word enzyme
11877-581: Was first done for lysozyme , an enzyme found in tears, saliva and egg whites that digests the coating of some bacteria; the structure was solved by a group led by David Chilton Phillips and published in 1965. This high-resolution structure of lysozyme marked the beginning of the field of structural biology and the effort to understand how enzymes work at an atomic level of detail. Enzymes can be classified by two main criteria: either amino acid sequence similarity (and thus evolutionary relationship) or enzymatic activity. Enzyme activity . An enzyme's name
11988-428: Was increased by twentyfold compared to reprogramming using mortal cells. The reactivation of hTERT, and subsequently telomerase , in human iPS cells has been used as an indication of pluripotency and reprogramming to an ES (embryonic stem) cell-like state when using mortal cells. Reprogrammed cells that do not express sufficient hTERT levels enter a quiescent state following a number of replications depending on
12099-427: Was shown to have drought-resistant-like properties, becoming upregulated during drought stress in the roots and leaves of Arabidopsis . NF-YA mutants have shown a loss of function and a hypersensitivity to drought-like conditions, and, in contrast, overexpression of NF-YA has resulted in drought resistance . The NF-YB family is, similar to the NF-YA subunit, variable in length, however, on average much smaller than
12210-455: Was the G residue towards the 5' end of the ATTGG. The other residues also listed were notable, but there is a split between two residues. This same experiment also yielded the same sequence as shown above when using a different oligonucleotide (R2) that contained an ATTGG core and flanked by 12 5' random nucleotides and 10 3' random nucleotides. Both these sequences are very similar and confirmed in multiple experiments. For sequences that flanked
12321-451: Was used later to refer to nonliving substances such as pepsin , and the word ferment was used to refer to chemical activity produced by living organisms. Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of experiments at the University of Berlin , he found that sugar was fermented by yeast extracts even when there were no living yeast cells in
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