A protein family is a group of evolutionarily-related proteins. In many cases a protein family has a corresponding gene family, in which each gene encodes a corresponding protein with a 1:1 relationship; the term protein family should not be confused with family. Proteins in a family descend from a common ancestor and have similar three-dimensional structures and significant sequence similarity; the most important of these is sequence similarity since it is the strictest indicator of homology and therefore the clearest indicator of common ancestry. There is a well developed framework for evaluating the significance of similarity between a group of sequences using sequence alignment methods. Proteins that do not share a common ancestor are unlikely to show statistically significant sequence similarity, making sequence alignment a powerful tool for identifying the members of protein families. Families are sometimes grouped together into larger clades called superfamilies based on structural and mechanistic similarity if there is no identifiable sequence homology.
Over 60,000 protein families have been defined, although ambiguity in the definition of protein family leads different researchers to wildly varying numbers. As with many biological terms, the use of protein family is somewhat context dependent. To distinguish between these situations, the term protein superfamily is used for distantly related proteins whose relatedness is not detectable by sequence similarity, but only from shared structural features. Other terms such as protein class, group and sub-family have been coined over the years, but all suffer similar ambiguities of usage. A common usage is. Hence a superfamily, such as the PA clan of proteases, has far lower sequence conservation than one of the families it contains, the C04 family, it is unlikely that an exact definition will be agreed and to it is up to the reader to discern how these terms are being used in a particular context.. The concept of protein family was conceived at a time when few protein structures or sequences were known.
Since that time, it was found that many proteins comprise multiple independent structural and functional units or domains. Due to evolutionary shuffling, different domains in a protein have evolved independently; this has led, to a focus on families of protein domains. A number of online resources are devoted to cataloging such domains. Regions of each protein have differing functional constraints. For example, the active site of an enzyme requires certain amino acid residues to be oriented in three dimensions. On the other hand, a protein–protein binding interface may consist of a large surface with constraints on the hydrophobicity or polarity of the amino acid residues. Functionally constrained regions of proteins evolve more than unconstrained regions such as surface loops, giving rise to discernible blocks of conserved sequence when the sequences of a protein family are compared; these blocks are most referred to as motifs, although many other terms are used. Again, a large number of online resources are devoted to cataloging protein motifs.
According to current consensus, protein families arise in two ways. Firstly, the separation of a parent species into two genetically isolated descendent species allows a gene/protein to independently accumulate variations in these two lineages; this results in a family of orthologous proteins with conserved sequence motifs. Secondly, a gene duplication may create a second copy of a gene; because the original gene is still able to perform its function, the duplicated gene is free to diverge and may acquire new functions. Certain gene/protein families in eukaryotes, undergo extreme expansions and contractions in the course of evolution, sometimes in concert with whole genome duplications; this expansion and contraction of protein families is one of the salient features of genome evolution, but its importance and ramifications are unclear. As the total number of sequenced proteins increases and interest expands in proteome analysis, there is an ongoing effort to organize proteins into families and to describe their component domains and motifs.
Reliable identification of protein families is critical to phylogenetic analysis, functional annotation, the exploration of diversity of protein function in a given phylogenetic branch. The Enzyme Function Initiative is using protein families and superfamilies as the basis for development of a sequence/structure-based strategy for large scale functional assignment of enzymes of unknown function; the algorithmic means for establishing protein families on a large scale are based on a notion of similarity. Most of the time the only similarity we have access to is sequence similarity. There are many biological databases that record examples of protein families and allow users to identify if newly identified proteins belong to a known family. Here are a few examples: Pfam - Prot
Protein Data Bank
The Protein Data Bank is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The data obtained by X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy, submitted by biologists and biochemists from around the world, are accessible on the Internet via the websites of its member organisations; the PDB is overseen by an organization called the Worldwide Protein Data Bank, wwPDB. The PDB is a key in areas such as structural genomics. Most major scientific journals, some funding agencies, now require scientists to submit their structure data to the PDB. Many other databases use protein structures deposited in the PDB. For example, SCOP and CATH classify protein structures, while PDBsum provides a graphic overview of PDB entries using information from other sources, such as Gene ontology. Two forces converged to initiate the PDB: 1) a small but growing collection of sets of protein structure data determined by X-ray diffraction.
In 1969, with the sponsorship of Walter Hamilton at the Brookhaven National Laboratory, Edgar Meyer began to write software to store atomic coordinate files in a common format to make them available for geometric and graphical evaluation. By 1971, one of Meyer's programs, SEARCH, enabled researchers to remotely access information from the database to study protein structures offline. SEARCH was instrumental in enabling networking, thus marking the functional beginning of the PDB; the Protein Data Bank was announced in October 1971 in Nature New Biology as a joint venture between Cambridge Crystallographic Data Centre, UK and Brookhaven National Laboratory, USA. Upon Hamilton's death in 1973, Tom Koeztle took over direction of the PDB for the subsequent 20 years. In January 1994, Joel Sussman of Israel's Weizmann Institute of Science was appointed head of the PDB. In October 1998, the PDB was transferred to the Research Collaboratory for Structural Bioinformatics; the new director was Helen M. Berman of Rutgers University.
In 2003, with the formation of the wwPDB, the PDB became an international organization. The founding members are PDBe, RCSB, PDBj; the BMRB joined in 2006. Each of the four members of wwPDB can act as deposition, data processing and distribution centers for PDB data; the data processing refers to the fact that annotate each submitted entry. The data are automatically checked for plausibility; the PDB database is updated weekly. The PDB holdings list is updated weekly; as of 17 October 2018, the breakdown of current holdings is as follows: 120,052 structures in the PDB have a structure factor file. 9,734 structures have an NMR restraint file. 3,486 structures in the PDB have a chemical shifts file. 2,531 structures in the PDB have a 3DEM map file deposited in EM Data BankThese data show that most structures are determined by X-ray diffraction, but about 10% of structures are now determined by protein NMR. When using X-ray diffraction, approximations of the coordinates of the atoms of the protein are obtained, whereas estimations of the distances between pairs of atoms of the protein are found through NMR experiments.
Therefore, the final conformation of the protein is obtained, in the latter case, by solving a distance geometry problem. A few proteins are determined by cryo-electron microscopy; the significance of the structure factor files, mentioned above, is that, for PDB structures determined by X-ray diffraction that have a structure file, the electron density map may be viewed. The data of such structures is stored on the "electron density server". In the past, the number of structures in the PDB has grown at an exponential rate, passing the 100 registered structures milestone in 1982, the 1,000 in 1993, the 10,000 in 1999, the 100,000 in 2014. However, since 2007, the rate of accumulation of new protein structures appears to have plateaued; the file format used by the PDB was called the PDB file format. This original format was restricted by the width of computer punch cards to 80 characters per line. Around 1996, the "macromolecular Crystallographic Information file" format, mmCIF, an extension of the CIF format started to be phased in.
MmCIF is now the master format for the PDB archive. An XML version of this format, called PDBML, was described in 2005; the structure files can be downloaded in any of these three formats. In fact, individual files are downloaded into graphics packages using web addresses: For PDB format files, use, e.g. http://www.pdb.org/pdb/files/4hhb.pdb.gz or http://pdbe.org/download/4hhb For PDBML files, use, e.g. http://www.pdb.org/pdb/files/4hhb.xml.gz or http://pdbe.org/pdbml/4hhbThe "4hhb" is the PDB identifier. Each structure published in PDB receives a four-character alphanumeric identifier, its PDB ID; the structure files may be viewed using one of several free and open source computer programs, including Jmol, Pymol, VMD, Rasmol. Other non-free, shareware programs
Pancreatic lipase family
Triglyceride lipases are a family of lipolytic enzymes that hydrolyse ester linkages of triglycerides. Lipases are distributed in animals and prokaryotes. At least three tissue-specific isozymes exist in higher vertebrates, pancreatic and gastric/lingual; these lipases are related to each other and to lipoprotein lipase, which hydrolyses triglycerides of chylomicrons and low density lipoproteins. The most conserved region in all these proteins is centred on a serine residue, shown to participate, with an histidine and an aspartic acid residue, in a charge relay system; such a region is present in lipases of prokaryotic origin and in lecithin-cholesterol acyltransferase, which catalyzes fatty acid transfer between phosphatidylcholine and cholesterol. Pancreatic lipase known as pancreatic triacylglycerol lipase or steapsin, is an enzyme secreted from the pancreas; as the primary lipase enzyme that hydrolyzes dietary fat molecules in the human digestive system, it is one of the main digestive enzymes, converting triglyceride substrates found in ingested oils to monoglycerides and free fatty acids.
Triacylglycerol + 2 H2O ⇌ 2-monoacylglycerol + 2 fatty acid anionsBile salts secreted from the liver and stored in gallbladder are released into the duodenum, where they coat and emulsify large fat droplets into smaller droplets, thus increasing the overall surface area of the fat, which allows the lipase to break apart the fat more effectively. The resulting monomers are moved by way of peristalsis along the small intestine to be absorbed into the lymphatic system by a specialized vessel called a lacteal; this protein belongs to the pancreatic lipase family. Unlike some pancreatic enzymes that are activated by proteolytic cleavage, pancreatic lipase is secreted in its final form. However, it becomes efficient only in the presence of colipase in the duodenum. In humans, pancreatic lipase is encoded by the PNLIP gene. LIPC LIPG LIPH LIPI LPL PLA1A PNLIP PNLIPRP1 PNLIPRP2 PNLIPRP3 Pancreatic lipase is secreted into the duodenum through the duct system of the pancreas, its concentration in serum is very low.
Under extreme disruption of pancreatic function, such as pancreatitis or pancreatic adenocarcinoma, the pancreas may begin to autolyse and release pancreatic enzymes including pancreatic lipase into serum. Thus, through measurement of serum concentration of pancreatic lipase, acute pancreatitis can be diagnosed. Lipase inhibitors such as orlistat can be used as a treatment for obesity. One peptide selected by phage display was found to inhibit pancreatic lipase. Orlistat Roussel A, Yang Y, Ferrato F, Verger R, Cambillau C, Lowe M. "Structure and activity of rat pancreatic lipase-related protein 2". J. Biol. Chem. 273: 32121–8. Doi:10.1074/jbc.273.48.32121. PMID 9822688. Crandall WV, Lowe ME. "Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles". J. Biol. Chem. 276: 12505–12. Doi:10.1074/jbc. M009986200. PMID 11278590. Freie AB, Ferrato F, Carrière F, Lowe ME. "Val-407 and Ile-408 in the beta5'-loop of pancreatic lipase mediate lipase-colipase interactions in the presence of bile salt micelles".
J. Biol. Chem. 281: 7793–800. Doi:10.1074/jbc. M512984200. PMC 3695395. PMID 16431912. Hegele RA, Ramdath DD, Ban MR, Carruthers MN, Carrington CV, Cao H. "Polymorphisms in PNLIP, encoding pancreatic lipase, associations with metabolic traits". J. Hum. Genet. 46: 320–4. Doi:10.1007/s100380170066. PMID 11393534. Chahinian H, Sias B, Carrière F. "The C-terminal domain of pancreatic lipase: functional and structural analogies with c2 domains". Curr. Protein Pept. Sci. 1: 91–103. PMID 12369922. Ranaldi S, Belle V, Woudstra M, Rodriguez J, Guigliarelli B, Sturgis J, Carriere F, Fournel A. "Lid opening and unfolding in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy". Biochemistry. 48: 630–8. Doi:10.1021/bi801250s. PMID 19113953. Grupe A, Li Y, Rowland C, Nowotny P, Hinrichs AL, Smemo S, Kauwe JS, Maxwell TJ, Cherny S, Doil L, Tacey K, van Luchene R, Myers A, Wavrant-De Vrièze F, Kaleem M, Hollingworth P, Jehu L, Foy C, Archer N, Hamilton G, Holmans P, Morris CM, Catanese J, Sninsky J, White TJ, Powell J, Hardy J, O'Donovan M, Lovestone S, Jones L, Morris JC, Thal L, Owen M, Williams J, Goate A.
"A scan of chromosome 10 identifies a novel locus showing strong association with late-onset Alzheimer disease". Am. J. Hum. Genet. 78: 78–88. Doi:10.1086/498851. PMC 1380225. PMID 16385451. Thomas A, Allouche M, Basyn F, Brasseur R, Kerfelec B. "Role of the lid hydrophobicity pattern in pancreatic lipase activity". J. Biol. Chem. 280: 40074–83. Doi:10.1074/jbc. M502123200. PMID 16179352. Van Tilbeurgh H, Egloff MP, Martinez C, Rugani N, Verger R, Cambillau C. "Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography". Nature. 362: 814–20. Bibcode:1993Natur.362..814V. Doi:10.1038/362814a0. PMID 8479519. Lessinger JM, Arzoglou P, Ramos P, Visvikis A, Parashou S, Calam D, Profilis C, Férard G. "Preparation and characterization of reference materials for human pancreatic lipase: BCR 693 and BCR 694". Clin. Chem. Lab. Med. 41: 169–76. Doi:10.1515/CCLM.2003.028. PMID 12667003. Colin DY, Deprez-Beauclair P, Allouche M, Brasseur R, Kerfelec B. "Exploring the active site cavity of human pancreatic lipase".
Biochem. Biophys. Res. Commun. 370: 394–8. Doi:10.1016/j.bbrc.2008.03.043. PMID 18353248
A chromosome is a deoxyribonucleic acid molecule with part or all of the genetic material of an organism. Most eukaryotic chromosomes include packaging proteins which, aided by chaperone proteins, bind to and condense the DNA molecule to prevent it from becoming an unmanageable tangle. Chromosomes are visible under a light microscope only when the cell is undergoing the metaphase of cell division. Before this happens, every chromosome is copied once, the copy is joined to the original by a centromere, resulting either in an X-shaped structure if the centromere is located in the middle of the chromosome or a two-arm structure if the centromere is located near one of the ends; the original chromosome and the copy are now called sister chromatids. During metaphase the X-shape structure is called a metaphase chromosome. In this condensed form chromosomes are easiest to distinguish and study. In animal cells, chromosomes reach their highest compaction level in anaphase during chromosome segregation.
Chromosomal recombination during meiosis and subsequent sexual reproduction play a significant role in genetic diversity. If these structures are manipulated incorrectly, through processes known as chromosomal instability and translocation, the cell may undergo mitotic catastrophe; this will make the cell initiate apoptosis leading to its own death, but sometimes mutations in the cell hamper this process and thus cause progression of cancer. Some use the term chromosome in a wider sense, to refer to the individualized portions of chromatin in cells, either visible or not under light microscopy. Others use the concept in a narrower sense, to refer to the individualized portions of chromatin during cell division, visible under light microscopy due to high condensation; the word chromosome comes from the Greek χρῶμα and σῶμα, describing their strong staining by particular dyes. The term was coined by von Waldeyer-Hartz, referring to the term chromatin, introduced by Walther Flemming; some of the early karyological terms have become outdated.
For example and Chromosom, both ascribe color to a non-colored state. The German scientists Schleiden, Virchow and Bütschli were among the first scientists who recognized the structures now familiar as chromosomes. In a series of experiments beginning in the mid-1880s, Theodor Boveri gave the definitive demonstration that chromosomes are the vectors of heredity, it is the second of these principles, so original. Wilhelm Roux suggested. Boveri was able to confirm this hypothesis. Aided by the rediscovery at the start of the 1900s of Gregor Mendel's earlier work, Boveri was able to point out the connection between the rules of inheritance and the behaviour of the chromosomes. Boveri influenced two generations of American cytologists: Edmund Beecher Wilson, Nettie Stevens, Walter Sutton and Theophilus Painter were all influenced by Boveri. In his famous textbook The Cell in Development and Heredity, Wilson linked together the independent work of Boveri and Sutton by naming the chromosome theory of inheritance the Boveri–Sutton chromosome theory.
Ernst Mayr remarks that the theory was hotly contested by some famous geneticists: William Bateson, Wilhelm Johannsen, Richard Goldschmidt and T. H. Morgan, all of a rather dogmatic turn of mind. Complete proof came from chromosome maps in Morgan's own lab; the number of human chromosomes was published in 1923 by Theophilus Painter. By inspection through the microscope, he counted 24 pairs, his error was copied by others and it was not until 1956 that the true number, 46, was determined by Indonesia-born cytogeneticist Joe Hin Tjio. The prokaryotes – bacteria and archaea – have a single circular chromosome, but many variations exist; the chromosomes of most bacteria, which some authors prefer to call genophores, can range in size from only 130,000 base pairs in the endosymbiotic bacteria Candidatus Hodgkinia cicadicola and Candidatus Tremblaya princeps, to more than 14,000,000 base pairs in the soil-dwelling bacterium Sorangium cellulosum. Spirochaetes of the genus Borrelia are a notable exception to this arrangement, with bacteria such as Borrelia burgdorferi, the cause of Lyme disease, containing a single linear chromosome.
Prokaryotic chromosomes have less sequence-based structure than eukaryotes. Bacteria have a one-point from which replication starts, whereas some archaea contain multiple replication origins; the genes in prokaryotes are organized in operons, do not contain introns, unlike eukaryotes. Prokaryotes do not possess nuclei. Instead, their DNA is organized into a structure called the nucleoid; the nucleoid occupies a defined region of the bacterial cell. This structure is, dynamic and is maintained and remodeled by the actions of a range of histone-like proteins, which associate with the bacterial chromosome. In archaea, the DNA in chromosomes is more organized, with the DNA packaged within structures similar to eukaryotic nucleosomes. Certain bacteria contain plasmids or other extrachromosomal DNA; these are circular structures in the cytoplasm that contain cellular DNA and play a role in horizontal gene transfer. In prokaryotes and viruses, the DNA is densely packed and organized.
A base pair is a unit consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA and RNA. Dictated by specific hydrogen bonding patterns, Watson–Crick base pairs allow the DNA helix to maintain a regular helical structure, subtly dependent on its nucleotide sequence; the complementary nature of this based-paired structure provides a redundant copy of the genetic information encoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing patterns that identify particular regulatory regions of genes. Intramolecular base pairs can occur within single-stranded nucleic acids.
This is important in RNA molecules, where Watson–Crick base pairs permit the formation of short double-stranded helices, a wide variety of non-Watson–Crick interactions allow RNAs to fold into a vast range of specific three-dimensional structures. In addition, base-pairing between transfer RNA and messenger RNA forms the basis for the molecular recognition events that result in the nucleotide sequence of mRNA becoming translated into the amino acid sequence of proteins via the genetic code; the size of an individual gene or an organism's entire genome is measured in base pairs because DNA is double-stranded. Hence, the number of total base pairs is equal to the number of nucleotides in one of the strands; the haploid human genome is estimated to be about 3.2 billion bases long and to contain 20,000–25,000 distinct protein-coding genes. A kilobase is a unit of measurement in molecular biology equal to 1000 base pairs of DNA or RNA; the total amount of related DNA base pairs on Earth is estimated at 5.0×1037 and weighs 50 billion tonnes.
In comparison, the total mass of the biosphere has been estimated to be as much as 4 TtC. Hydrogen bonding is the chemical interaction. Appropriate geometrical correspondence of hydrogen bond donors and acceptors allows only the "right" pairs to form stably. DNA with high GC-content is more stable than DNA with low GC-content. But, contrary to popular belief, the hydrogen bonds do not stabilize the DNA significantly; the larger nucleobases and guanine, are members of a class of double-ringed chemical structures called purines. Purines are complementary only with pyrimidines: pyrimidine-pyrimidine pairings are energetically unfavorable because the molecules are too far apart for hydrogen bonding to be established. Purine-pyrimidine base-pairing of AT or GC or UA results in proper duplex structure; the only other purine-pyrimidine pairings would be AC and GT and UG. The GU pairing, with two hydrogen bonds, does occur often in RNA. Paired DNA and RNA molecules are comparatively stable at room temperature, but the two nucleotide strands will separate above a melting point, determined by the length of the molecules, the extent of mispairing, the GC content.
Higher GC content results in higher melting temperatures. On the converse, regions of a genome that need to separate — for example, the promoter regions for often-transcribed genes — are comparatively GC-poor. GC content and melting temperature must be taken into account when designing primers for PCR reactions; the following DNA sequences illustrate pair double-stranded patterns. By convention, the top strand is written from the 5' end to the 3' end. A base-paired DNA sequence: ATCGATTGAGCTCTAGCG TAGCTAACTCGAGATCGCThe corresponding RNA sequence, in which uracil is substituted for thymine in the RNA strand: AUCGAUUGAGCUCUAGCG UAGCUAACUCGAGAUCGC Chemical analogs of nucleotides can take the place of proper nucleotides and establish non-canonical base-pairing, leading to errors in DNA replication and DNA transcription; this is due to their isosteric chemistry. One common mutagenic base analog is 5-bromouracil, which resembles thymine but can base-pair to guanine in its enol form. Other chemicals, known as DNA intercalators, fit into the gap between adjacent bases on a single strand and induce frameshift mutations by "masquerading" as a base, causing the DNA replication machinery to skip or insert additional nucleotides at the intercalated site.
Most intercalators are known or suspected carcinogens. Examples include ethidium acridine. An unnatural base pair is a designed subunit of DNA, created in a laboratory and does not occur in nature. DNA sequences have been described which use newly created nucleobases to form a third base pair, in addition to the two ba
Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, transporting molecules from one location to another. Proteins differ from one another in their sequence of amino acids, dictated by the nucleotide sequence of their genes, which results in protein folding into a specific three-dimensional structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are considered to be proteins and are called peptides, or sometimes oligopeptides; the individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residues in a protein is defined by the sequence of a gene, encoded in the genetic code.
In general, the genetic code specifies 20 standard amino acids. Shortly after or during synthesis, the residues in a protein are chemically modified by post-translational modification, which alters the physical and chemical properties, stability and the function of the proteins. Sometimes proteins have non-peptide groups attached, which can be called prosthetic groups or cofactors. Proteins can work together to achieve a particular function, they associate to form stable protein complexes. Once formed, proteins only exist for a certain period and are degraded and recycled by the cell's machinery through the process of protein turnover. A protein's lifespan covers a wide range, they can exist for years with an average lifespan of 1 -- 2 days in mammalian cells. Abnormal or misfolded proteins are degraded more either due to being targeted for destruction or due to being unstable. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in every process within cells.
Many proteins are enzymes that are vital to metabolism. Proteins have structural or mechanical functions, such as actin and myosin in muscle and the proteins in the cytoskeleton, which form a system of scaffolding that maintains cell shape. Other proteins are important in cell signaling, immune responses, cell adhesion, the cell cycle. In animals, proteins are needed in the diet to provide the essential amino acids that cannot be synthesized. Digestion breaks the proteins down for use in the metabolism. Proteins may be purified from other cellular components using a variety of techniques such as ultracentrifugation, precipitation and chromatography. Methods used to study protein structure and function include immunohistochemistry, site-directed mutagenesis, X-ray crystallography, nuclear magnetic resonance and mass spectrometry. Most proteins consist of linear polymers built from series of up to 20 different L-α- amino acids. All proteinogenic amino acids possess common structural features, including an α-carbon to which an amino group, a carboxyl group, a variable side chain are bonded.
Only proline differs from this basic structure as it contains an unusual ring to the N-end amine group, which forces the CO–NH amide moiety into a fixed conformation. The side chains of the standard amino acids, detailed in the list of standard amino acids, have a great variety of chemical structures and properties; the amino acids in a polypeptide chain are linked by peptide bonds. Once linked in the protein chain, an individual amino acid is called a residue, the linked series of carbon and oxygen atoms are known as the main chain or protein backbone; the peptide bond has two resonance forms that contribute some double-bond character and inhibit rotation around its axis, so that the alpha carbons are coplanar. The other two dihedral angles in the peptide bond determine the local shape assumed by the protein backbone; the end with a free amino group is known as the N-terminus or amino terminus, whereas the end of the protein with a free carboxyl group is known as the C-terminus or carboxy terminus.
The words protein and peptide are a little ambiguous and can overlap in meaning. Protein is used to refer to the complete biological molecule in a stable conformation, whereas peptide is reserved for a short amino acid oligomers lacking a stable three-dimensional structure. However, the boundary between the two is not well defined and lies near 20–30 residues. Polypeptide can refer to any single linear chain of amino acids regardless of length, but implies an absence of a defined conformation. Proteins can interact with many types of molecules, including with other proteins, with lipids, with carboyhydrates, with DNA, it has been estimated. Smaller bacteria, such as Mycoplasma or spirochetes contain fewer molecules, on the order of 50,000 to 1 million. By contrast, eukaryotic cells are larger and thus contain much more pro
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme inhibition; the quantity or concentration of an enzyme can be expressed in molar amounts, as with any other chemical, or in terms of activity in enzyme units. Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified; the SI unit is the katal, 1 katal = 1 mol s−1, but this is an excessively large unit. A more practical and used value is enzyme unit = 1 μmol min−1. 1 U corresponds to 16.67 nanokatals. Enzyme activity as given in katal refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can be given as that of certain standardized substrates, such as gelatin measured in gelatin digesting units, or milk proteins measured in milk clotting units; the units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively.
1 GDU equals 1.5 MCU. An increased amount of substrate will increase the rate of reaction with enzymes, however once past a certain point, the rate of reaction will level out because the amount of active sites available has stayed constant; the specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein. Specific activity gives a measurement of enzyme purity in the mixture, it is the micro moles of product formed by an enzyme in a given amount of time under given conditions per milligram of total proteins. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein; the SI unit is katal/kg. Specific activity is a measure of enzyme processivity, at a specific substrate concentration, is constant for a pure enzyme. An active site titration process can be done for the elimination of errors arising from differences in cultivation batches and/or misfolded enzyme and similar issues.
This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should be expressed as μmol min−1 mg−1 active enzyme. If the molecular weight of the enzyme is known, the turnover number, or μmol product per second per μmol of active enzyme, can be calculated from the specific activity; the turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second. The rate of a reaction is the concentration of substrate disappearing per unit time; the % purity is 100% ×. The impure sample has lower specific activity because some of the mass is not enzyme. If the specific activity of 100% pure enzyme is known an impure sample will have a lower specific activity, allowing purity to be calculated. All enzyme assays measure either the consumption of production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways.
Biochemists study enzyme-catalysed reactions using four types of experiments: Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient; the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains constant over time and the reaction rate changes slowly. Rates are measured for a short period after the attainment of the quasi-steady state by monitoring the accumulation of product with time; because the measurements are carried out for a short period and because of the large excess of substrate, the approximation that the amount of free substrate is equal to the amount of the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being free from complications such as back-reaction and enzyme degradation, it is therefore by far the most used type of experiment in enzyme kinetics. Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time.
The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. Progress curve experiments were used in the early period of enzyme kinetics, but are less common now. Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period; these experiments are more difficult to perform than either of the above two classes because they require specialist techniques or rapid mixing. Relaxation experiments. In these experiments, an equilibrium mixture of enzyme and product is perturbed, for instance by a temperature, pressure or pH jump, the return to equilibrium is monitored; the analysis of these experiments requires consideration of the reversible reaction. Moreover, relaxation experiments are insensitive to mechanistic details and are thus not used for mechanism identification, although they can be under appropriate c