Ureases ( EC 3.5.1.5 ), functionally, belong to the superfamily of amidohydrolases and phosphotriesterases. Ureases are found in numerous bacteria , fungi , algae , plants, and some invertebrates , as well as in soils, as a soil enzyme . They are nickel-containing metalloenzymes of high molecular weight.
126-400: These enzymes catalyze the hydrolysis of urea into carbon dioxide and ammonia : The hydrolysis of urea occurs in two stages. In the first stage, ammonia and carbamic acid are produced. The carbamate spontaneously and rapidly hydrolyzes to ammonia and carbonic acid . Urease activity increases the pH of its environment as ammonia is produced, which is basic. Its activity
252-457: A carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid . Urea serves an important role in the cellular metabolism of nitrogen -containing compounds by animals and is the main nitrogen-containing substance in the urine of mammals . Urea is Neo-Latin , from French urée , from Ancient Greek οὖρον ( oûron ) 'urine', itself from Proto-Indo-European *h₂worsom . It
378-487: 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
504-488: A class of chemical compounds that share the same functional group, a carbonyl group attached to two organic amine residues: R R N−C(=O)−NR R , where R , R , R and R groups are hydrogen (–H), organyl or other groups. Examples include carbamide peroxide , allantoin , and hydantoin . Ureas are closely related to biurets and related in structure to amides , carbamates , carbodiimides , and thiocarbamides . More than 90% of world industrial production of urea
630-489: 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
756-448: A crystal in 1926 by Sumner, using acetone solvation and centrifuging. Modern biochemistry has increased its demand for urease. Jack bean meal , watermelon seeds , and pea seeds have all proven useful sources of urease. 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
882-477: 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
1008-412: A muscle loss of 0.67 gram. In aquatic organisms the most common form of nitrogen waste is ammonia, whereas land-dwelling organisms convert the toxic ammonia to either urea or uric acid . Urea is found in the urine of mammals and amphibians , as well as some fish. Birds and saurian reptiles have a different form of nitrogen metabolism that requires less water, and leads to nitrogen excretion in
1134-464: 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
1260-547: A range of condensation products , including cyanuric acid (CNOH) 3 , guanidine HNC(NH 2 ) 2 , and melamine . In aqueous solution, urea slowly equilibrates with ammonium cyanate. This elimination reaction cogenerates isocyanic acid , which can carbamylate proteins, in particular the N-terminal amino group, the side chain amino of lysine , and to a lesser extent the side chains of arginine and cysteine . Each carbamylation event adds 43 daltons to
1386-439: 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
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#17327982203681512-456: A reaction that produces ammonia from urea. This increases the pH (reduces the acidity) of the stomach environment around the bacteria. Similar bacteria species to H. pylori can be identified by the same test in animals such as apes , dogs , and cats (including big cats ). Amino acids from ingested food (or produced from catabolism of muscle protein) that are used for the synthesis of proteins and other biological substances can be oxidized by
1638-581: A reaction with nitric acid to make urea nitrate , a high explosive that is used industrially and as part of some improvised explosive devices . Urea is used in Selective Non-Catalytic Reduction (SNCR) and Selective Catalytic Reduction (SCR) reactions to reduce the NO x pollutants in exhaust gases from combustion from diesel , dual fuel, and lean-burn natural gas engines. The BlueTec system, for example, injects
1764-611: A reverse protonation scheme, where a protonated form of the His ligand plays the role of the general acid and the Ni2-bound water is already in the deprotonated state. The mechanism follows the same path, with the general base omitted (as there is no more need for it) and His donating its proton to form the ammonia molecule, which is then released from the enzyme. While the majority of the His ligands and bound water will not be in their active forms (protonated and deprotonated, respectively,) it
1890-410: A solid highly soluble in water (545 g/L at 25 °C), urea is much easier and safer to handle and store than the more irritant , caustic and hazardous ammonia ( NH 3 ), so it is the reactant of choice. Trucks and cars using these catalytic converters need to carry a supply of diesel exhaust fluid , also sold as AdBlue , a solution of urea in water. Urea in concentrations up to 10 M
2016-443: A source of nitrogen (N) and is an important raw material for the chemical industry . In 1828, Friedrich Wöhler discovered that urea can be produced from inorganic starting materials, which was an important conceptual milestone in chemistry. This showed for the first time that a substance previously known only as a byproduct of life could be synthesized in the laboratory without biological starting materials, thereby contradicting
2142-451: 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
2268-449: 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
2394-406: A stronger odor than fresh urine. The cycling of and excretion of urea by the kidneys is a vital part of mammalian metabolism. Besides its role as carrier of waste nitrogen, urea also plays a role in the countercurrent exchange system of the nephrons , that allows for reabsorption of water and critical ions from the excreted urine . Urea is reabsorbed in the inner medullary collecting ducts of
2520-442: 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
2646-763: A total of 12 active sites. It plays an essential function for survival, neutralizing gastric acid by allowing urea to enter into periplasm via a proton-gated urea channel . The presence of urease is used in the diagnosis of Helicobacter species. All bacterial ureases are solely cytoplasmic, except for those in Helicobacter pylori , which along with its cytoplasmic activity, has external activity with host cells. In contrast, all plant ureases are cytoplasmic. Fungal and plant ureases are made up of identical subunits (~90 kDa each), most commonly assembled as trimers and hexamers. For example, jack bean urease has two structural and one catalytic subunit. The α subunit contains
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#17327982203682772-464: A water-based urea solution into the exhaust system. Ammonia ( NH 3 ) produced by the hydrolysis of urea reacts with nitrogen oxides ( NO x ) and is converted into nitrogen gas ( N 2 ) and water within the catalytic converter. The conversion of noxious NO x to innocuous N 2 is described by the following simplified global equation: When urea is used, a pre-reaction (hydrolysis) occurs to first convert it to ammonia: Being
2898-457: 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
3024-838: 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
3150-427: Is a colorless, odorless solid, highly soluble in water, and practically non-toxic ( LD 50 is 15 g/kg for rats). Dissolved in water, it is neither acidic nor alkaline . The body uses it in many processes, most notably nitrogen excretion . The liver forms it by combining two ammonia molecules ( NH 3 ) with a carbon dioxide ( CO 2 ) molecule in the urea cycle . Urea is widely used in fertilizers as
3276-449: Is a common byproduct of the metabolism of nitrogenous compounds. Ammonia is smaller, more volatile, and more mobile than urea. If allowed to accumulate, ammonia would raise the pH in cells to toxic levels. Therefore, many organisms convert ammonia to urea, even though this synthesis has a net energy cost. Being practically neutral and highly soluble in water, urea is a safe vehicle for the body to transport and excrete excess nitrogen. Urea
3402-813: Is a powerful protein denaturant as it disrupts the noncovalent bonds in the proteins. This property can be exploited to increase the solubility of some proteins. A mixture of urea and choline chloride is used as a deep eutectic solvent (DES), a substance similar to ionic liquid . When used in a deep eutectic solvent, urea gradually denatures the proteins that are solubilized. Urea in concentrations up to 8 M can be used to make fixed brain tissue transparent to visible light while still preserving fluorescent signals from labeled cells. This allows for much deeper imaging of neuronal processes than previously obtainable using conventional one photon or two photon confocal microscopes. Urea-containing creams are used as topical dermatological products to promote rehydration of
3528-421: 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
3654-699: Is a recommended preparation procedure. However, cyanate will build back up to significant levels within a few days. Alternatively, adding 25–50 mM ammonium chloride to a concentrated urea solution decreases formation of cyanate because of the common ion effect . Urea is readily quantified by a number of different methods, such as the diacetyl monoxime colorimetric method, and the Berthelot reaction (after initial conversion of urea to ammonia via urease). These methods are amenable to high throughput instrumentation, such as automated flow injection analyzers and 96-well micro-plate spectrophotometers. Ureas describes
3780-607: Is a weak base, with a p K b of 13.9. When combined with strong acids, it undergoes protonation at oxygen to form uronium salts. It is also a Lewis base , forming metal complexes of the type [M(urea) 6 ] . Urea reacts with malonic esters to make barbituric acids . Molten urea decomposes into ammonium cyanate at about 152 °C, and into ammonia and isocyanic acid above 160 °C: Heating above 160 °C yields biuret NH 2 CONHCONH 2 and triuret NH 2 CONHCONHCONH 2 via reaction with isocyanic acid: At higher temperatures it converts to
3906-526: Is also the cause of peptic ulcers with its manifestation in 55–68% reported cases. This was confirmed by decreased ulcer bleeding and ulcer reoccurrence after eradication of the pathogen . In the stomach there is an increase in pH of the mucosal lining as a result of urea hydrolysis , which prevents movement of hydrogen ions between gastric glands and gastric lumen . In addition, the high ammonia concentrations have an effect on intercellular tight junctions increasing permeability and also disrupting
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4032-414: Is controlled by the antidiuretic hormone , to create hyperosmotic urine — i.e., urine with a higher concentration of dissolved substances than the blood plasma . This mechanism is important to prevent the loss of water, maintain blood pressure , and maintain a suitable concentration of sodium ions in the blood plasma. The equivalent nitrogen content (in grams ) of urea (in mmol ) can be estimated by
4158-437: 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
4284-409: Is destined for use as a nitrogen-release fertilizer . Urea has the highest nitrogen content of all solid nitrogenous fertilizers in common use. Therefore, it has a low transportation cost per unit of nitrogen nutrient . The most common impurity of synthetic urea is biuret , which impairs plant growth. Urea breaks down in the soil to give ammonium ions ( NH + 4 ). The ammonium is taken up by
4410-492: Is equivalent to a fused γ-β-α organization. The Helicobacter "α" is equivalent to a fusion of the normal bacterial γ-β subunits, while its "β" subunit is equivalent to the normal bacterial α. The three-chain organization is likely ancestral. The k cat / K m of urease in the processing of urea is 10 times greater than the rate of the uncatalyzed elimination reaction of urea . There are many reasons for this observation in nature. The proximity of urea to active groups in
4536-749: 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
4662-502: Is located in the α (alpha) subunits . It is a bis-μ-hydroxo dimeric nickel center, with an interatomic distance of ~3.5 Å. > The Ni(II) pair are weakly antiferromagnetically coupled. X-ray absorption spectroscopy (XAS) studies of Canavalia ensiformis (jack bean), Klebsiella aerogenes and Sporosarcina pasteurii (formerly known as Bacillus pasteurii ) confirm 5–6 coordinate nickel ions with exclusively O/N ligation, including two imidazole ligands per nickel. Urea substrate
4788-666: Is no longer in widespread use. The blood urea nitrogen (BUN) test is a measure of the amount of nitrogen in the blood that comes from urea. It is used as a marker of renal function , though it is inferior to other markers such as creatinine because blood urea levels are influenced by other factors such as diet, dehydration, and liver function. Urea has also been studied as an excipient in Drug-coated Balloon (DCB) coating formulation to enhance local drug delivery to stenotic blood vessels. Urea, when used as an excipient in small doses (~3 μg/mm ) to coat DCB surface
4914-510: Is often attributed to the French chemist Hilaire Rouelle as well as William Cruickshank . Boerhaave used the following steps to isolate urea: In 1828, the German chemist Friedrich Wöhler obtained urea artificially by treating silver cyanate with ammonium chloride . This was the first time an organic compound was artificially synthesized from inorganic starting materials, without
5040-476: 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
5166-422: Is often measured as an indicator of the health of microbial communities. In the absence of plants, urease activity in soil is generally attributed to heterotrophic microorganisms, although it has been demonstrated that some chemoautotrophic ammonium oxidizing bacteria are capable of growth on urea as a sole source of carbon, nitrogen, and energy. The inhibition of urease is a significant goal in agriculture because
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5292-418: 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
5418-462: 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
5544-501: Is produced from microbial urease during urea hydrolysis , as this increases the surrounding environments pH from roughly 6.5 to 9. The resultant alkalinization results in stone crystallization . In humans the microbial urease, Proteus mirabilis , is the most common in infection induced urinary stones. Studies have shown that Helicobacter pylori along with cirrhosis of the liver cause hepatic encephalopathy and hepatic coma . Helicobacter pylori release microbial ureases into
5670-466: Is proposed to displace aquo ligands . Water molecules located towards the opening of the active site form a tetrahedral cluster that fills the cavity site through hydrogen bonds . Some amino acid residues are proposed to form mobile flap of the site, which gate for the substrate. Cysteine residues are common in the flap region of the enzymes, which have been determined not to be essential in catalysis, although involved in positioning other key residues in
5796-404: 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
5922-420: Is synthesized in the body of many organisms as part of the urea cycle , either from the oxidation of amino acids or from ammonia . In this cycle, amino groups donated by ammonia and L - aspartate are converted to urea, while L - ornithine , citrulline , L - argininosuccinate , and L - arginine act as intermediates. Urea production occurs in the liver and is regulated by N -acetylglutamate . Urea
6048-403: 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
6174-437: Is then dissolved into the blood (in the reference range of 2.5 to 6.7 mmol/L) and further transported and excreted by the kidney as a component of urine . In addition, a small amount of urea is excreted (along with sodium chloride and water) in sweat . In water, the amine groups undergo slow displacement by water molecules, producing ammonia, ammonium ions , and bicarbonate ions . For this reason, old, stale urine has
6300-790: 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:
6426-614: 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
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#17327982203686552-511: 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
6678-432: The skin . Urea 40% is indicated for psoriasis , xerosis , onychomycosis , ichthyosis , eczema , keratosis , keratoderma , corns, and calluses . If covered by an occlusive dressing , 40% urea preparations may also be used for nonsurgical debridement of nails . Urea 40% "dissolves the intercellular matrix" of the nail plate. Only diseased or dystrophic nails are removed, as there is no effect on healthy portions of
6804-495: The trigonal planar angle of 120° and the tetrahedral angle of 109.5°. In solid urea, the oxygen center is engaged in two N–H–O hydrogen bonds . The resulting hydrogen-bond network is probably established at the cost of efficient molecular packing: The structure is quite open, the ribbons forming tunnels with square cross-section. The carbon in urea is described as sp hybridized, the C-N bonds have significant double bond character, and
6930-624: The Ciurli/Mangani mechanism and the other two is that it incorporates a nitrogen , oxygen bridging urea that is attacked by a bridging hydroxide . Bacterial ureases are often the mode of pathogenesis for many medical conditions. They are associated with hepatic encephalopathy / Hepatic coma , infection stones, and peptic ulceration. Infection induced urinary stones are a mixture of struvite (MgNH 4 PO 4 •6H 2 O) and carbonate apatite [Ca 10 (PO 4 )6•CO 3 ]. These polyvalent ions are soluble but become insoluble when ammonia
7056-400: 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
7182-493: The active site along with the correct orientation of urea allow hydrolysis to occur rapidly. Urea alone is very stable due to the resonance forms it can adopt. The stability of urea is understood to be due to its resonance energy, which has been estimated at 30–40 kcal/mol. This is because the zwitterionic resonance forms all donate electrons to the carbonyl carbon making it less of an electrophile making it less reactive to nucleophilic attack. The active site of ureases
7308-437: 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
7434-404: The active site appropriately. In Sporosarcina pasteurii urease, the flap was found in the open conformation, while its closed conformation is apparently needed for the reaction. When compared, the α subunits of Helicobacter pylori urease and other bacterial ureases align with the jack bean ureases. The binding of urea to the active site of urease has not been observed. One mechanism for
7560-560: The active site, it is composed of 840 amino acids per molecule (90 cysteines), its molecular mass without Ni(II) ions amounting to 90.77 kDa. The mass of the hexamer with the 12 nickel ions is 545.34 kDa. Other examples of homohexameric structures of plant ureases are those of soybean, pigeon pea and cotton seeds enzymes. It is important to note, that although composed of different types of subunits, ureases from different sources extending from bacteria to plants and fungi exhibit high homology of amino acid sequences. The single plant urease chain
7686-502: 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
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#17327982203687812-444: The advent of dialysis . It is the classical pre-dialysis era description of crystallized urea deposits over the skin of patients with prolonged kidney failure and severe uremia. Urea was first noticed by Herman Boerhaave in the early 18th century from evaporates of urine. In 1773, Hilaire Rouelle obtained crystals containing urea from human urine by evaporating it and treating it with alcohol in successive filtrations. This method
7938-429: The amino acid sequence). 840 amino acids per molecule, of which 90 are cysteine residues. The optimum pH is 7.4 and optimum temperature is 60 °C. Substrates include urea and hydroxyurea . Bacterial ureases are composed of three distinct subunits, one large catalytic (α 60–76kDa) and two small (β 8–21 kDa, γ 6–14 kDa) commonly forming (αβγ) 3 trimers stoichiometry with a 2-fold symmetric structure (note that
8064-407: 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
8190-578: 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
8316-417: The binding of urea to the active site is achieved via a hydrogen-bonding network, orienting the substrate into the catalytic cavity. Urea binds to the five-coordinated nickel (Ni1) with the carbonyl oxygen atom. It approaches the six-coordinated nickel (Ni2) with one of its amino groups and subsequently bridges the two nickel centers. The binding of the urea carbonyl oxygen atom to Ni1 is stabilized through
8442-502: 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
8568-528: The body as an alternative source of energy, yielding urea and carbon dioxide . The oxidation pathway starts with the removal of the amino group by a transaminase ; the amino group is then fed into the urea cycle . The first step in the conversion of amino acids into metabolic waste in the liver is removal of the alpha-amino nitrogen, which produces ammonia . Because ammonia is toxic, it is excreted immediately by fish, converted into uric acid by birds, and converted into urea by mammals. Ammonia ( NH 3 )
8694-500: The carbonyl oxygen is relatively basic. Urea's high aqueous solubility reflects its ability to engage in extensive hydrogen bonding with water. By virtue of its tendency to form porous frameworks, urea has the ability to trap many organic compounds. In these so-called clathrates , the organic "guest" molecules are held in channels formed by interpenetrating helices composed of hydrogen-bonded urea molecules. In this way, urea-clathrates have been well investigated for separations. Urea
8820-460: The carbonyl oxygens of Ala and Ala enhance the basicity of the NH 2 groups and allow for binding to Ni2. Therefore, in this proposed mechanism, the positioning of urea in the active site is induced by the structural features of the active site residues which are positioned to act as hydrogen-bond donors in the vicinity of Ni1 and as acceptors in the vicinity of Ni2. The main structural difference between
8946-460: The catalysis of this reaction by urease was proposed by Blakely and Zerner. It begins with a nucleophilic attack by the carbonyl oxygen of the urea molecule onto the 5-coordinate Ni (Ni-1). A weakly coordinated water ligand is displaced in its place. A lone pair of electrons from one of the nitrogen atoms on the Urea molecule creates a double bond with the central carbon, and the resulting NH 2 of
9072-403: The catalytic function is disabled. These include entomotoxicity, inhibition of fungi, neurotoxicity in mammals, promotion of endocytosis and inflammatory eicosanoid production in mammals, and induction of chemotaxis in bacteria. These activities may be part of a defense mechanism. Urease insect-toxicity was originally noted in canatoxin, an orthologous isoform of jack bean urease. Digestion of
9198-428: The chemical composition of the pure substance. In the evolved procedure, urea was precipitated as urea nitrate by adding strong nitric acid to urine. To purify the resulting crystals, they were dissolved in boiling water with charcoal and filtered. After cooling, pure crystals of urea nitrate form. To reconstitute the urea from the nitrate, the crystals are dissolved in warm water, and barium carbonate added. The water
9324-471: 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
9450-407: The conversion factor 0.028 g/mmol. Furthermore, 1 gram of nitrogen is roughly equivalent to 6.25 grams of protein , and 1 gram of protein is roughly equivalent to 5 grams of muscle tissue. In situations such as muscle wasting , 1 mmol of excessive urea in the urine (as measured by urine volume in litres multiplied by urea concentration in mmol/L) roughly corresponds to
9576-425: 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
9702-425: The coordinated substrate interacts with a nearby positively charged group. Blakeley and Zerner proposed this nearby group to be a Carboxylate ion , although deprotonated carboxylates are negatively charged. A hydroxide ligand on the six coordinate Ni is deprotonated by a base. The carbonyl carbon is subsequently attacked by the electronegative oxygen. A pair of electrons from the nitrogen-carbon double bond returns to
9828-444: 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
9954-422: The detection of urease to be used as a diagnostic to detect presence of pathogens. Urease-positive pathogens include: A wide range of urease inhibitors of different structural families are known. Inhibition of urease is not only of interest to agriculture, but also to medicine as pathogens like H. pylori produce urease as a survival mechanism. Known structural classes of inhibitors include: First isolated as
10080-433: 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"
10206-592: 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
10332-483: 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,
10458-422: 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
10584-403: 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
10710-427: 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
10836-473: The form of uric acid. Tadpoles excrete ammonia, but shift to urea production during metamorphosis . Despite the generalization above, the urea pathway has been documented not only in mammals and amphibians, but in many other organisms as well, including birds, invertebrates , insects, plants, yeast , fungi , and even microorganisms . Urea can be irritating to skin, eyes, and the respiratory tract. Repeated or prolonged contact with urea in fertilizer form on
10962-411: The gastric mucous membrane of the stomach. Urea is found naturally in the environment and is also artificially introduced, comprising more than half of all synthetic nitrogen fertilizers used globally. Heavy use of urea is thought to promote eutrophication , despite the observation that urea is rapidly transformed by microbial ureases, and thus usually does not persist. Environmental urease activity
11088-474: The image above gives the structure of the asymmetric unit, one-third of the true biological assembly), they are cysteine-rich enzymes, resulting in the enzyme molar masses between 190 and 300kDa. An exceptional urease is obtained from Helicobacter sp.. These are composed of two subunits, α(26–31 kDa)-β(61–66 kDa). These subunits form a supramolecular (αβ) 12 dodecameric complex. of repeating α-β subunits, each coupled pair of subunits has an active site, for
11214-469: 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. Urea 50 g/L ethanol ~4 g/L acetonitrile Urea , also called carbamide (because it is a diamide of carbonic acid ), is an organic compound with chemical formula CO(NH 2 ) 2 . This amide has two amino groups (– NH 2 ) joined by
11340-519: The involvement of living organisms. The results of this experiment implicitly discredited vitalism , the theory that the chemicals of living organisms are fundamentally different from those of inanimate matter. This insight was important for the development of organic chemistry . His discovery prompted Wöhler to write triumphantly to Jöns Jakob Berzelius : In fact, his second sentence was incorrect. Ammonium cyanate [NH 4 ] [OCN] and urea CO(NH 2 ) 2 are two different chemicals with
11466-408: The mass of the protein, which can be observed in protein mass spectrometery . For this reason, pure urea solutions should be freshly prepared and used, as aged solutions may develop a significant concentration of cyanate (20 mM in 8 M urea). Dissolving urea in ultrapure water followed by removing ions (i.e. cyanate) with a mixed-bed ion-exchange resin and storing that solution at 4 °C
11592-400: The mechanism of urease and is based primarily on the different roles of the two nickel ions in the active site. One of which binds and activates urea, the other nickel ion binds and activates the nucleophilic water molecule. With regards to this proposal, urea enters the active site cavity when the mobile ‘flap’ (which allows for the entrance of urea into the active site) is open. Stability of
11718-474: 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
11844-431: The mobile flap region of the enzyme. As this histidine ligand is part of the mobile flap, binding of the urea substrate for catalysis closes this flap over the active site and with the addition of the hydrogen bonding pattern to urea from other ligands in the pocket, speaks to the selectivity of the urease enzyme for urea. The mechanism proposed by Ciurli and Mangani is one of the more recent and currently accepted views of
11970-409: The nail. This drug (as carbamide peroxide ) is also used as an earwax removal aid. Urea has also been studied as a diuretic . It was first used by Dr. W. Friedrich in 1892. In a 2010 study of ICU patients, urea was used to treat euvolemic hyponatremia and was found safe, inexpensive, and simple. Like saline , urea has been injected into the uterus to induce abortion , although this method
12096-402: The nephrons, thus raising the osmolarity in the medullary interstitium surrounding the thin descending limb of the loop of Henle , which makes the water reabsorb. By action of the urea transporter 2 , some of this reabsorbed urea eventually flows back into the thin descending limb of the tubule, through the collecting ducts, and into the excreted urine. The body uses this mechanism, which
12222-399: The nitrogen and neutralizes the charge on it, while the now 4-coordinate carbon assumes an intermediate tetrahedral orientation. The breakdown of this intermediate is then helped by a sulfhydryl group of a cysteine located near the active site. A hydrogen bonds to one of the nitrogen atoms, breaking its bond with carbon, and releasing an NH 3 molecule. Simultaneously, the bond between
12348-488: The oxygen and the 6-coordinate nickel is broken. This leaves a carbamate ion coordinated to the 5-coordinate Ni, which is then displaced by a water molecule, regenerating the enzyme. The carbamate produced then spontaneously degrades to produce another ammonia and carbonic acid . The mechanism proposed by Hausinger and Karplus attempts to revise some of the issues apparent in the Blakely and Zerner pathway, and focuses on
12474-436: The peptide identified a 10-kDa portion most responsible for this effect, termed jaburetox. An analogous portion from the soybean urease is named soyuretox. Studies on insects show that the entire protein is toxic without needing any digestion, however. Nevertheless, the "uretox" peptides, being more concentrated in toxicity, show promise as biopesticides . Many gastrointestinal or urinary tract pathogens produce urease, enabling
12600-412: The plant through its roots. In some soils, the ammonium is oxidized by bacteria to give nitrate ( NO − 3 ), which is also a nitrogen-rich plant nutrient. The loss of nitrogenous compounds to the atmosphere and runoff is wasteful and environmentally damaging so urea is sometimes modified to enhance the efficiency of its agricultural use. Techniques to make controlled-release fertilizers that slow
12726-433: The positions of the side chains making up the urea-binding pocket. From the crystal structures from K. aerogenes urease, it was argued that the general base used in the Blakely mechanism, His, was too far away from the Ni2-bound water to deprotonate in order to form the attacking hydroxide moiety. In addition, the general acidic ligand required to protonate the urea nitrogen was not identified. Hausinger and Karplus suggests
12852-528: 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
12978-488: The protonation state of His Nԑ. Additionally, the conformational change from the open to closed state of the mobile flap generates a rearrangement of Ala carbonyl group in such a way that its oxygen atom points to Ni2. The Ala and Ala are now oriented in a way that their carbonyl groups act as hydrogen-bond acceptors towards NH 2 group of urea, thus aiding its binding to Ni2. Urea is a very poor chelating ligand due to low Lewis base character of its NH 2 groups. However
13104-539: The rapid breakdown of urea-based fertilizers is wasteful and environmentally damaging. Phenyl phosphorodiamidate and N -( n -butyl)thiophosphoric triamide are two such inhibitors. By promoting the formation of calcium carbonate , ureases are potentially useful for biomineralization -inspired processes. Notably, microbiologically induced formation of calcium carbonate can be used in making bioconcrete . In addition to acting as an enzyme, some ureases (especially plant ones) have additional effects that persist even when
13230-406: 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
13356-733: 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
13482-462: The release of nitrogen include the encapsulation of urea in an inert sealant, and conversion of urea into derivatives such as urea-formaldehyde compounds, which degrade into ammonia at a pace matching plants' nutritional requirements. Urea is a raw material for the manufacture of formaldehyde based resins , such as UF, MUF, and MUPF, used mainly in wood-based panels, for instance, particleboard , fiberboard , OSB, and plywood . Urea can be used in
13608-459: The runoff from fertilized land may play a role in the increase of toxic blooms. The substance decomposes on heating above melting point, producing toxic gases, and reacts violently with strong oxidants, nitrites, inorganic chlorides, chlorites and perchlorates, causing fire and explosion. Urea was first discovered in urine in 1727 by the Dutch scientist Herman Boerhaave , although this discovery
13734-436: The same empirical formula CON 2 H 4 , which are in chemical equilibrium heavily favoring urea under standard conditions . Regardless, with his discovery, Wöhler secured a place among the pioneers of organic chemistry. Uremic frost was first described in 1865 by Harald Hirschsprung , the first Danish pediatrician in 1870 who also described the disease that carries his name in 1886. Uremic frost has become rare since
13860-410: 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
13986-469: The skin may cause dermatitis . High concentrations in the blood can be damaging. Ingestion of low concentrations of urea, such as are found in typical human urine , are not dangerous with additional water ingestion within a reasonable time-frame. Many animals (e.g. camels , rodents or dogs) have a much more concentrated urine which may contain a higher urea amount than normal human urine. Urea can cause algal blooms to produce toxins, and its presence in
14112-419: The stomach. The urease hydrolyzes urea to produce ammonia and carbonic acid . As the bacteria are localized to the stomach ammonia produced is readily taken up by the circulatory system from the gastric lumen . This results in elevated ammonia levels in the blood, a condition known as hyperammonemia ; eradication of Helicobacter pylori show marked decreases in ammonia levels. Helicobacter pylori
14238-412: 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
14364-519: 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
14490-405: 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
14616-399: 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
14742-438: 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
14868-436: The widely held doctrine of vitalism , which stated that only living organisms could produce the chemicals of life. The structure of the molecule of urea is O=C(−NH 2 ) 2 . The urea molecule is planar when in a solid crystal because of sp hybridization of the N orbitals. It is non-planar with C 2 symmetry when in the gas phase or in aqueous solution, with C–N–H and H–N–H bond angles that are intermediate between
14994-486: 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
15120-439: Was aided by Carl Wilhelm Scheele 's discovery that urine treated by concentrated nitric acid precipitated crystals. Antoine François, comte de Fourcroy and Louis Nicolas Vauquelin discovered in 1799 that the nitrated crystals were identical to Rouelle's substance and invented the term "urea." Berzelius made further improvements to its purification and finally William Prout , in 1817, succeeded in obtaining and determining
15246-520: Was awarded the Nobel prize in chemistry in 1946. The crystal structure of urease was first solved by P. A. Karplus in 1995. A 1984 study focusing on urease from jack bean found that the active site contains a pair of nickel centers. In vitro activation also has been achieved with manganese and cobalt in place of nickel. Lead salts are inhibiting . The molecular weight is either 480 kDa or 545 kDa for jack-bean urease (calculated mass from
15372-433: Was calculated that approximately 0.3% of total urease enzyme would be active at any one time. While logically, this would imply that the enzyme is not very efficient, contrary to established knowledge, usage of the reverse protonation scheme provides an advantage in increased reactivity for the active form, balancing out the disadvantage. Placing the His ligand as an essential component in the mechanism also takes into account
15498-581: 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
15624-410: Was first identified in 1876 by Frédéric Alphonse Musculus as a soluble ferment. In 1926, James B. Sumner , showed that urease is a protein by examining its crystallized form. Sumner's work was the first demonstration that a protein can function as an enzyme and led eventually to the recognition that most enzymes are in fact proteins. Urease was the first enzyme crystallized. For this work, Sumner
15750-449: Was found to form crystals that increase drug transfer without adverse toxic effects on vascular endothelial cells . Urea labeled with carbon-14 or carbon-13 is used in the urea breath test , which is used to detect the presence of the bacterium Helicobacter pylori ( H. pylori ) in the stomach and duodenum of humans, associated with peptic ulcers . The test detects the characteristic enzyme urease , produced by H. pylori , by
15876-457: 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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