101-432: Beta-lactamases ( β-lactamases ) are enzymes ( EC 3.5.2.6 ) produced by bacteria that provide multi-resistance to beta-lactam antibiotics such as penicillins , cephalosporins , cephamycins , monobactams and carbapenems ( ertapenem ), although carbapenems are relatively resistant to beta-lactamase. Beta-lactamase provides antibiotic resistance by breaking the antibiotics ' structure. These antibiotics all have
202-487: 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
303-489: 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
404-511: A type of enzyme rather than being like an enzyme, but even in 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
505-452: A chromogenic cephalosporin substrate which changes color from yellow to red upon beta-lactamase mediated hydrolysis. Extended spectrum beta lactamase (ESBL) screening can be performed using disk-diffusion. Cefpodoxime, ceftazidime, aztreonam, cefotaxime, and/or ceftriaxone discs are used. Beta-lactamases are ancient bacterial enzymes. Metallo β-lactamases ("class B") are all structurally similar to RNase Z and may have evolved from it. Of
606-685: A class C carbapenemase has been described. Plasmid-mediated IMP-type carbapenemases (IMP stands for active-on-imipenem), 19 varieties of which are currently known, became established in Japan in the 1990s both in enteric gram-negative organisms and in Pseudomonas and Acinetobacter species. IMP enzymes spread slowly to other countries in the Far East, were reported from Europe in 1997, and have been found in Canada and Brazil. A second growing family of carbapenemases,
707-435: A common element in their molecular structure: a four-atom ring known as a beta-lactam (β-lactam) ring. Through hydrolysis , the enzyme lactamase breaks the β-lactam ring open, deactivating the molecule's antibacterial properties. Beta-lactamases produced by gram-negative bacteria are usually secreted, especially when antibiotics are present in the environment. The structure of a Streptomyces serine β-lactamase (SBLs)
808-474: A first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions in high-fidelity mammalian polymerases. Similar proofreading mechanisms are also found in RNA polymerase , aminoacyl tRNA synthetases and ribosomes . Conversely, some enzymes display enzyme promiscuity , having broad specificity and acting on
909-774: A limited number of geographic sites. PER-1 in isolates in Turkey, France, and Italy; VEB-1 and VEB-2 in strains from Southeast Asia; and GES-1, GES-2, and IBC-2 in isolates from South Africa, France, and Greece. PER-1 is also common in multiresistant acinetobacter species in Korea and Turkey. Some of these enzymes are found in Enterobacteriaceae as well, whereas other uncommon ESBLs (such as BES-1, IBC-1, SFO-1, and TLA-1) have been found only in Enterobacteriaceae. While ESBL-producing organisms were previously associated with hospitals and institutional care, these organisms are now increasingly found in
1010-461: A new threat, since they confer resistance to 7-alpha-methoxy-cephalosporins ( cephamycins ) such as cefoxitin or cefotetan but are not affected by commercially available β-lactamase inhibitors, and can, in strains with loss of outer membrane porins, provide resistance to carbapenems. Members of this family commonly express β-lactamases (e.g., TEM-3, TEM-4, and SHV-2 ) which confer resistance to expanded-spectrum (extended-spectrum) cephalosporins. In
1111-464: 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
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#17330846500011212-439: 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
1313-403: A recent comparison of ciprofloxacin and imipenem for bacteremia involving an ESBL-producing K. pneumoniae , imipenem produced the better outcome There have been few clinical studies to define the optimal therapy for infections caused by ESBL producing Pseudomonas aeruginosa strains. In 1957, amid concern about allergic reactions to penicillin-containing antibiotics, a beta-lactamase
1414-743: A similar overall structure. The SHV-1 beta-lactamase is most commonly found in K. pneumoniae and is responsible for up to 20% of the plasmid-mediated ampicillin resistance in this species. ESBLs in this family also have amino acid changes around the active site, most commonly at positions 238 or 238 and 240. More than 60 SHV varieties are known. SHV-5 and SHV-12 are among the most common. The initials stand for "sulfhydryl reagent variable". These enzymes were named for their greater activity against cefotaxime than other oxyimino-beta-lactam substrates (e.g., ceftazidime , ceftriaxone , or cefepime ). Rather than arising by mutation, they represent examples of plasmid acquisition of beta-lactamase genes normally found on
1515-451: 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
1616-446: A steady level inside the cell. For example, NADPH is regenerated through the pentose phosphate pathway and S -adenosylmethionine by methionine adenosyltransferase . This continuous regeneration means that small amounts of coenzymes can be used very intensively. For example, the human body turns over its own weight in ATP each day. As with all catalysts, enzymes do not alter the position of
1717-560: A strain carbapenem-resistant as well. Strains with IMP-, VIM-, and OXA -type carbapenemases usually remain susceptible. Resistance to non-beta-lactam antibiotics is common in strains making any of these enzymes, such that alternative options for non-beta-lactam therapy need to be determined by direct susceptibility testing. Resistance to fluoroquinolones and aminoglycosides is especially high. For infections caused by ESBL-producing Escherichia coli or Klebsiella species, treatment with imipenem or meropenem has been associated with
1818-449: A tRNase Z, and that the B3 MBL activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are thought to have evolved through PNGM-1 activity. Subclasses B1 and B3 has been further subdivided. Serine beta-lactamases (classes A, C, and D) appear to have evolved from DD -transpeptidases , which are penicillin-binding proteins involved in cell wall biosynthesis, and as such are one of
1919-442: 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
2020-457: 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
2121-838: 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
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#17330846500012222-421: 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
2323-605: Is a specific type of β-lactamase, showing specificity for penicillins , again by hydrolysing the β-lactam ring. Molecular weights of the various penicillinases tend to cluster near 50 kilodaltons. Penicillinase was the first β-lactamase to be identified. It was first isolated by Abraham and Chain in 1940 from E. coli (which are gram-negative) even before penicillin entered clinical use, but penicillinase production quickly spread to bacteria that previously did not produce it or produced it only rarely. Penicillinase-resistant beta-lactams such as methicillin were developed, but there
2424-434: Is common, but both drugs show an inoculum effect, with diminished susceptibility as the size of the inoculum is increased from 10 to 10 organisms. Strains with some CTX-M –type and OXA -type ESBLs are resistant to cefepime on testing, despite the use of a standard inoculum. Although the inhibitor-resistant TEM variants are resistant to inhibition by clavulanic acid and sulbactam , thereby showing clinical resistance to
2525-437: 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
2626-749: 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
2727-549: Is given by 1BSG . The alpha-beta fold ( InterPro : IPR012338 ) resembles that of a DD -transpeptidase , from which the enzyme is thought to have evolved. β-lactam antibiotics bind to DD -transpeptidases to inhibit bacterial cell wall biosynthesis. Serine β-lactamases are grouped by sequence similarity into types A, C, and D. The other type of beta-lactamase is of the metallo type ("type B"). Metallo-beta-lactamases (MBLs) need metal ion(s) (1 or 2 Zn ions) on their active site for their catalytic activities. The structure of
2828-469: Is no longer a primarily European epidemiology, it is found in northern parts of America often and should be tested for with complex UTI's. AmpC type β-lactamases are commonly isolated from extended-spectrum cephalosporin-resistant gram-negative bacteria. AmpC β-lactamases (also termed class C or group 1) are typically encoded on the chromosome of many gram-negative bacteria including Citrobacter , Serratia and Enterobacter species where its expression
2929-542: Is now widespread resistance to even these. Among gram-negative bacteria, the emergence of resistance to extended-spectrum cephalosporins has been a major concern. It appeared initially in a limited number of bacterial species ( E. cloacae , C. freundii , S. marcescens , and P. aeruginosa ) that could mutate to hyperproduce their chromosomal class C β-lactamase. A few years later, resistance appeared in bacterial species not naturally producing AmpC enzymes ( K. pneumoniae , Salmonella spp., P. mirabilis ) due to
3030-473: Is often derived from its substrate or the chemical reaction it catalyzes, with the word ending in -ase . Examples are lactase , alcohol dehydrogenase and DNA polymerase . Different enzymes that catalyze the same chemical reaction are called isozymes . The International Union of Biochemistry and Molecular Biology have developed a nomenclature for enzymes, the EC numbers (for "Enzyme Commission") . Each enzyme
3131-418: 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
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3232-462: 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
3333-404: 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
3434-752: Is tested (in vitro) as susceptible. Associated resistance to aminoglycosides and trimethoprim - sulfamethoxazole , as well as high frequency of co-existence of fluoroquinolone resistance, creates problems. Beta-lactamase inhibitors such as clavulanate , sulbactam , and tazobactam in vitro inhibit most ESBLs, but the clinical effectiveness of beta-lactam/beta-lactamase inhibitor combinations cannot be relied on consistently for therapy. Cephamycins ( cefoxitin and cefotetan ) are not hydrolyzed by majority of ESBLs, but are hydrolyzed by associated AmpC-type β-lactamase. Also, β-lactam/β-lactamase inhibitor combinations may not be effective against organisms that produce AmpC-type β-lactamase. Sometimes these strains decrease
3535-403: 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
3636-531: Is the most commonly encountered beta-lactamase in gram-negative bacteria . Up to 90% of ampicillin resistance in E. coli is due to the production of TEM-1. Also responsible for the ampicillin and penicillin resistance that is seen in H. influenzae and N. gonorrhoeae in increasing numbers. Although TEM-type beta-lactamases are most often found in E. coli and K. pneumoniae , they are also found in other species of gram-negative bacteria with increasing frequency. The amino acid substitutions responsible for
3737-456: Is theorized to have evolved on chromosomes and moved to plasmids on at least two separate occasions. The "β" ( beta ) refers to the nitrogen 's position on the second carbon in the ring. Lactam is a blend of lactone (from the Latin lactis , milk , since lactic acid was isolated from soured milk) and amide . The suffix -ase , indicating an enzyme, is derived from diastase (from
3838-910: Is up to 10% in the VIM family, 15% in the IMP family, and 70% between VIM and IMP. Enzymes of both the families, nevertheless, are similar. Both are integron-associated, sometimes within plasmids. Both hydrolyse all β-lactams except monobactams, and evade all β-lactam inhibitors. The VIM enzymes are among the most widely distributed MBLs, with >40 VIM variants having been reported. Biochemical and biophysical studies revealed that VIM variants have only small variations in their kinetic parameters but substantial differences in their thermal stabilities and inhibition profiles. The OXA group of β-lactamases occur mainly in Acinetobacter species and are divided into two clusters. OXA carbapenemases hydrolyse carbapenems very slowly in vitro , and
3939-790: 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:
4040-583: Is usually inducible ; it may also occur on Escherichia coli but is not usually inducible, although it can be hyperexpressed. AmpC type β-lactamases may also be carried on plasmids. AmpC β-lactamases, in contrast to ESBLs, hydrolyse broad and extended-spectrum cephalosporins (cephamycins as well as to oxyimino-β-lactams) but are not typically inhibited by the β-lactamase inhibitors clavulanic acid and tazobactam , whereas avibactam can maintain inhibitory activity against this class of β-lactamases. AmpC-type β-lactamase organisms are often clinically grouped through
4141-611: The DNA polymerases ; here the holoenzyme is the complete complex containing all the subunits needed for activity. Coenzymes are small organic molecules that can be loosely or tightly bound to an enzyme. Coenzymes transport chemical groups from one enzyme to another. Examples include NADH , NADPH and adenosine triphosphate (ATP). Some coenzymes, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP), and tetrahydrofolate (THF), are derived from vitamins . These coenzymes cannot be synthesized by
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4242-482: The Greek diastasis , "separation"), the first enzyme discovered in 1833 by Payen and Persoz. 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 the enzyme converts the substrates into different molecules known as products . Almost all metabolic processes in
4343-553: The New Delhi metallo-beta-lactamase 1 is given by 6C89 . It resembles a RNase Z , from which it is thought to have evolved. The two types of beta-lactamases work on the basis of the two basic mechanisms of opening the β-lactam ring. The SBLs are similar in structure and mechanistically to the β-lactam target penicillin-binding proteins (PBPs) which are necessary for cell wall building and modifying. SBLs and PBPs both covalently change an active site serine residue. The difference between
4444-639: 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 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
4545-425: The cephamycins ( cefoxitin , cefotetan ) but resistance to the third-generation cephalosporins and to aztreonam . Moreover, one should suspect these strains when treatment with these agents for gram-negative infections fails despite reported in vitro susceptibility. Once an ESBL-producing strain is detected, the laboratory should report it as "resistant" to all penicillins, cephalosporins, and aztreonam, even if it
4646-417: The extended-spectrum beta lactamase (ESBL) phenotype cluster around the active site of the enzyme and change its configuration, allowing access to oxyimino-beta-lactam substrates. Opening the active site to beta-lactam substrates also typically enhances the susceptibility of the enzyme to β-lactamase inhibitors, such as clavulanic acid. Single amino acid substitutions at positions 104, 164, 238, and 240 produce
4747-511: 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
4848-458: The 1980s they have since the early 2000s spread and are the now the predominant ESBL type in the world. They are generally clustred into five groups based on sequencing homologies; CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25. CTX-M-15 (belonging to the CTX-M-1 cluster) is the most prevalent CTX-M-gene. An example of beta-lactamase CTX-M-15, along with IS Ecp1 , has been found to have transposed onto
4949-619: The ESBL phenotype, but ESBLs with the broadest spectrum usually have more than a single amino acid substitution. Based upon different combinations of changes, currently 140 TEM-type enzymes have been described. TEM-10, TEM-12, and TEM-26 are among the most common in the United States. The term TEM comes from the name of the Athenian patient (Temoniera) from which the isolate was recovered in 1963. SHV-1 shares 68 percent of its amino acids with TEM-1 and has
5050-505: The PBPs and SBLs is that the latter generates free enzyme and inactive antibiotic by the very quick hydrolysis of the acyl-enzyme intermediate. The MBLs use the Zn ions to activate a binding site water molecule for the hydrolysis of the β-lactam ring. Zinc chelators have recently been investigated as metallo-β-lactamase inhibitors, as they are often able to restore carbapenem susceptibility. Penicillinase
5151-502: The United States and UK), most probably due to the large number of tourists travelling the globe, who may have picked up the strain from the environment, as strains containing the NDM-1 gene have been found in environmental samples in India. NDM have several variants which share different properties. In general, an isolate is suspected to be an ESBL producer when it shows in vitro susceptibility to
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#17330846500015252-552: The VIM family, was reported from Italy in 1999 and now includes 10 members, which have a wide geographic distribution in Europe, South America, and the Far East and have been found in the United States. VIM-1 was discovered in P. aeruginosa in Italy in 1996; since then, VIM-2 - now the predominant variant - was found repeatedly in Europe and the Far East; VIM-3 and -4 are minor variants of VIM-2 and -1, respectively. Amino acid sequence diversity
5353-579: The acronym, "SPACE": Serratia , Pseudomonas or Proteus , Acinetobacter , Citrobacter , and Enterobacter . Carbapenems are famously stable to AmpC β-lactamases and extended-spectrum-β-lactamases. Carbapenemases are a diverse group of β-lactamases that are active not only against the oxyimino-cephalosporins and cephamycins but also against the carbapenems. Aztreonam is stable to the metallo-β-lactamases, but many IMP and VIM producers are resistant, owing to other mechanisms. Carbapenemases were formerly believed to derive only from classes A, B, and D, but
5454-437: 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
5555-502: 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
5656-407: 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
5757-578: 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
5858-469: The best outcomes in terms of survival and bacteriologic clearance. Cefepime and piperacillin/tazobactam have been less successful. Ceftriaxone , cefotaxime , and ceftazidime have failed even more often, despite the organism's susceptibility to the antibiotic in vitro . Several reports have documented failure of cephamycin therapy as a result of resistance due to porin loss. Some patients have responded to aminoglycoside or quinolone therapy, but, in
5959-462: The beta-lactam—beta lactamase inhibitor combinations of amoxicillin - clavulanate ( Co-amoxiclav ), ticarcillin - clavulanate , and ampicillin/sulbactam , they remain susceptible to inhibition by tazobactam and subsequently the combination of piperacillin/tazobactam . AmpC-producing strains are typically resistant to oxyimino-beta lactams and to cephamycins and are susceptible to carbapenems ; however, diminished porin expression can make such
6060-502: 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
6161-471: 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
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#17330846500016262-638: The chromosome of Klebsiella pneumoniae ATCC BAA-2146. The initials stand for "Cefotaxime-Munich". OXA beta-lactamases were long recognized as a less common but also plasmid-mediated beta-lactamase variety that could hydrolyze oxacillin and related anti-staphylococcal penicillins. These beta-lactamases differ from the TEM and SHV enzymes in that they belong to molecular class D and functional group 2d. The OXA-type beta-lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and cloxacillin and
6363-504: The chromosome of Kluyvera species, a group of rarely pathogenic commensal organisms. These enzymes are not very closely related to TEM or SHV beta-lactamases in that they show only approximately 40% identity with these two commonly isolated beta-lactamases. More than 172 CTX-M enzymes are currently known. Despite their name, a few are more active on ceftazidime than cefotaxime . They are widely described among species of Enterobacteriaceae , mainly E. coli and K. pneumoniae . Detected in
6464-450: The community. CTX-M-15-positive E. coli are a cause of community-acquired urinary infections in the UK, and tend to be resistant to all oral β-lactam antibiotics, as well as quinolones and sulfonamides . Treatment options may include nitrofurantoin , fosfomycin , mecillinam and chloramphenicol . In desperation, once-daily ertapenem or gentamicin injections may also be used. Although
6565-425: 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
6666-433: 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"
6767-587: The enzyme urease was a pure protein and crystallized it; he did likewise for the enzyme catalase in 1937. The conclusion that pure proteins can be enzymes was definitively demonstrated by John Howard Northrop and Wendell Meredith Stanley , who worked on the digestive enzymes pepsin (1930), trypsin and chymotrypsin . These three scientists were awarded the 1946 Nobel Prize in Chemistry. The discovery that enzymes could be crystallized eventually allowed their structures to be solved by x-ray crystallography . This
6868-483: 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,
6969-482: The enzyme since expression demands appropriate migration of an insertion sequence. CcrA was known before imipenem was introduced, and producers have shown little subsequent increase. Originally described from New Delhi in 2009, this gene is now widespread in Escherichia coli and Klebsiella pneumoniae from India and Pakistan. As of mid-2010, NDM-1 carrying bacteria have been introduced to other countries (including
7070-403: 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
7171-427: 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
7272-874: The expression of outer membrane proteins, rendering them resistant to cephamycins. In vivo studies have yielded mixed results against ESBL-producing K. pneumoniae . ( Cefepime , a fourth-generation cephalosporin, has demonstrated in vitro stability in the presence of many ESBL/AmpC strains.) Currently, carbapenems are, in general, regarded as the preferred agent for treatment of infections due to ESBL-producing organisms. Carbapenems are resistant to ESBL-mediated hydrolysis and exhibit excellent in vitro activity against strains of Enterobacteriaceae expressing ESBLs. Strains producing only ESBLs are susceptible to cephamycins and carbapenems in vitro and show little if any inoculum effect with these agents. For organisms producing TEM and SHV type ESBLs, apparent in vitro sensitivity to cefepime and to piperacillin/tazobactam
7373-533: The fact that they are poorly inhibited by clavulanic acid . Amino acid substitutions in OXA enzymes can also give the ESBL phenotype. While most ESBLs have been found in E. coli , K. pneumoniae , and other Enterobacteriaceae , the OXA-type ESBLs have been found mainly in P. aeruginosa . OXA-type ESBLs have been found mainly in Pseudomonas aeruginosa isolates from Turkey and France. The OXA beta-lactamase family
7474-560: The high MICs seen for some Acinetobacter hosts (>64 mg/L) may reflect secondary mechanisms. They are sometimes augmented in clinical isolates by additional resistance mechanisms, such as impermeability or efflux. OXA carbapenemases also tend to have a reduced hydrolytic efficiency towards penicillins and cephalosporins. A few class A enzymes, most noted the plasmid-mediated KPC enzymes, are effective carbapenemases as well. Ten variants, KPC-2 through KPC-11 are known, and they are distinguished by one or two amino acid substitutions (KPC-1
7575-493: 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. Ceftizoxime Ceftizoxime is a third-generation cephalosporin available for parenteral administration . Unlike other third-generation cephalosporins, the whole C-3 side chain in ceftizoxime has been removed to prevent deactivation by hydrolytic enzymes . It rather resembles cefotaxime in its properties, but
7676-484: The inhibitor-resistant TEM variants are resistant to inhibition by clavulanic acid and sulbactam , thereby showing clinical resistance to the beta-lactam—lactamase inhibitor combinations of amoxicillin - clavulanate ( co-amoxiclav ), ticarcillin - clavulanate ( co-ticarclav ), and ampicillin/sulbactam , they normally remain susceptible to inhibition by tazobactam and subsequently the combination of piperacillin/tazobactam , although resistance has been described. This
7777-627: The inhibitor-resistant β-lactamases are not ESBLs, they are often discussed with ESBLs because they are also derivatives of the classical TEM- or SHV-type enzymes. These enzymes were at first given the designation IRT for inhibitor-resistant TEM β-lactamase; however, all have subsequently been renamed with numerical TEM designations. There are at least 19 distinct inhibitor-resistant TEM β-lactamases. Inhibitor-resistant TEM β-lactamases have been found mainly in clinical isolates of E. coli , but also some strains of K. pneumoniae , Klebsiella oxytoca , P. mirabilis , and Citrobacter freundii . Although
7878-429: The main targets of beta-lactam antibiotics. These three classes show undetectable sequence similarity with each other, but can still be compared using structural homology. Groups A and D are sister taxa and group C diverged before A and D. These serine-based enzymes, like the group B betalactamases, are of ancient origin and are theorized to have evolved about two billion years ago. The OXA group (in class D) in particular
7979-717: The mid-1980s, this new group of enzymes, the extended-spectrum β-lactamases (ESBLs), was detected (first detected in 1979). The prevalence of ESBL-producing bacteria have been gradually increasing in acute care hospitals. The prevalence in the general population varies between countries, e.g. approximately 6% in Germany and France, 13% in Saudi Arabia, and 63% in Egypt. ESBLs are beta-lactamases that hydrolyze extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include cefotaxime , ceftriaxone , and ceftazidime , as well as
8080-468: The mixture. He named the enzyme that brought about the fermentation of sucrose " zymase ". In 1907, he received the Nobel Prize in Chemistry for "his discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named according to the reaction they carry out: the suffix -ase is combined with the name of the substrate (e.g., lactase is the enzyme that cleaves lactose ) or to
8181-689: The oxyimino-monobactam aztreonam . Thus ESBLs confer multi-resistance to these antibiotics and related oxyimino-beta lactams. In typical circumstances, they derive from genes for TEM-1, TEM-2, or SHV-1 by mutations that alter the amino acid configuration around the active site of these β-lactamases. A broader set of β-lactam antibiotics are susceptible to hydrolysis by these enzymes. An increasing number of ESBLs not of TEM or SHV lineage have recently been described. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in
8282-531: The polio vaccine, which used penicillin as a preservative. However, some patients developed allergies to neutrapen. The Albany Hospital removed it from its formulary in 1960, only two years after adding it, citing lack of use. Some researchers continued to use it in experiments on penicillin resistance as late as 1972. It was voluntarily withdrawn from the American market by 3M Pharmaceuticals in 1997. Beta-lactamase enzymatic activity can be detected using nitrocefin ,
8383-528: 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
8484-545: The production of TEM- or SHV-type ESBLs (extended spectrum beta lactamases). Characteristically, such resistance has included oxyimino- (for example ceftizoxime , cefotaxime , ceftriaxone , and ceftazidime , as well as the oxyimino-monobactam aztreonam ), but not 7-alpha-methoxy-cephalosporins ( cephamycins ; in other words, cefoxitin and cefotetan ); has been blocked by inhibitors such as clavulanate , sulbactam or tazobactam and did not involve carbapenems and temocillin . Chromosomal-mediated AmpC β-lactamases represent
8585-406: 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
8686-733: 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
8787-410: 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
8888-412: 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
8989-519: 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
9090-405: 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
9191-399: 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
9292-425: The three subclasses B1, B2, and B3, B1 and B2 are theorized to have evolved about one billion years ago , while B3 seems to have arisen independently, possibly before the divergence of the gram-positive and gram-negative eubacteria about two billion years ago. PNGM-1 (Papua New Guinea Metallo-β-lactamase-1) has both metallo-β-lactamase (MBL) and tRNase Z activities, suggesting that PNGM-1 is thought to have evolved from
9393-445: The treatment of ESBL-producing organisms are extremely limited. Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant (primarily ertapenem -resistant) isolates have recently been reported. ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins . However, treatment with such antibiotics has been associated with high failure rates. TEM-1
9494-438: 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
9595-486: 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
9696-583: Was first detected in 1996 in North Carolina , USA. A 2010 publication indicated that KPC producing Enterobacteriaceae were becoming common in the United States. The first class C carbapenemase was described in 2006 and was isolated from a virulent strain of Enterobacter aerogenes . It is carried on a plasmid, pYMG-1, and is therefore transmissible to other bacterial strains. In general, these are of little clinical significance. CcrA (CfiA). Its gene occurs in ca. 1–3% of B. fragilis isolates, but fewer produce
9797-581: 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
9898-704: Was originally created as a phenotypic rather than a genotypic group for a few beta-lactamases that had a specific hydrolysis profile. Therefore, there is as little as 20% sequence homology among some of the members of this family. However, recent additions to this family show some degree of homology to one or more of the existing members of the OXA beta-lactamase family. Some confer resistance predominantly to ceftazidime, but OXA-17 confers greater resistance to cefotaxime and cefepime than it does resistance to ceftazidime. Other plasmid-mediated ESBLs, such as PER, VEB, GES, and IBC beta-lactamases, have been described but are uncommon and have been found mainly in P. aeruginosa and at
9999-585: Was re-sequenced in 2008 and found to be 100% homologous to published sequences of KPC-2). KPC-1 was found in North Carolina, KPC-2 in Baltimore and KPC-3 in New York. They have only 45% homology with SME and NMC/IMI enzymes and, unlike them, can be encoded by self-transmissible plasmids. As of February 2009, the class A Klebsiella pneumoniae carbapenemase ( KPC ) globally has been the most common carbapenemase, and
10100-422: Was sold as an antidote under the brand name neutrapen. It was theorized that the breakdown of penicillin by the enzyme would treat the allergic reaction. While it was not useful in acute anaphylactic shock, it showed positive results in cases of urticaria and joint pain suspected to be caused by penicillin allergy. Its use was proposed in pediatric cases where penicillin allergy was discovered upon administration of
10201-451: Was used later to refer to nonliving substances such as pepsin , and the word ferment was used to refer to chemical activity produced by living organisms. Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of experiments at the University of Berlin , he found that sugar was fermented by yeast extracts even when there were no living yeast cells in
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