As shown in Intermediate I of the figure, three potential acidic amino acid residues interact with the N-terminus of the substrate in a fashion that is yet to be determined. The carbonyl and amide groups of the scissile peptide bond interact with the first metal ion, M1, in addition to His178 and His79, respectively. M1 and Glu204 activate a water molecule to prepare it nucleophilic attack at the carbonyl carbon of the scissile peptide bond.
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The structure of the glmS ribozyme was first determined by X-ray crystallography in 2006. The tertiary structure of this RNA is characterized by three coaxial stacked helices, packed side-by-side. The ribozyme core contains a double pseudoknotted structure, which places the central helix P2.1 such that the scissile phosphate is nestled by the major groove. The major groove of the adjacent helix P2.2 is involved in metabolite binding and the scissile phosphate is attached to the 5' end of the helix.
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The mechanism for MMP-3 is a variation on a larger theme seen in all matrix metalloproteinases. In the active site, a water molecule is coordinated to a glutamate residue (Glu202) and one of the zinc ions present in the catalytic domain. First, the coordinated water molecule performs a nucleophilic attack on the peptide substrate's scissile carbon while the glutamate simultaneously abstracts a proton from the water molecule. The abstracted proton is then removed from the glutamate by the nitrogen of the scissile amide.
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In molecular biology, a scissile bond is a covalent chemical bond that can be broken by an enzyme. Examples would be the cleaved bond in the self-cleaving hammerhead ribozyme or the peptide bond of a substrate cleaved by a peptidase.
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Keto-ACE was used as the basis for the design of ketomethylene derivates. Its analogues contain a ketomethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedron transition state of the proteolytic reaction at the active site.
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While a number of different mechanisms for aspartyl proteases have been proposed, the most widely accepted is a general acid-base mechanism involving coordination of a water molecule between the two highly conserved aspartate residues. One aspartate activates the water by abstracting a proton, enabling the water to perform a nucleophilic attack on the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate stabilized by hydrogen-bonding with the second aspartic acid. Rearrangement of this intermediate leads to protonation of the scissile amide which results in the splitting of the substrate peptide into two product peptides.
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The cleavage of peptide bonds by OmpT is also dependent on the presence of bound lipopolysaccharide (LPS). When LPS is not present, the peptide binds too deeply within the active site, and the water cannot reach the carbonyl for its nucleophilic attack of the scissile bond.
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Cyanopyrrolidines have two key interactions to the DPP-4 complex: 1\. Nitrile in the position of the scissile bond of the peptidic substrate that is important for high potency. The nitrile group forms reversible covalent bonds with the catalytically active serine hydroxyl (Ser630), i.e. cyanopyrrolidines are competitive inhibitors with slow dissociation kinetics. 2\.
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When hydrolyzing a substrate, DPEP1 goes through a tetrahedral intermediate, after which the bridging solvent attacks the face of the carbonyl carbon of the scissile peptide bond.Thoden, James B., Ricardo Marti- Arbona, Frank M. Raushel, and Hazel M. Holden. "High-Resolution X-Ray Structure of Isoaspartyl Dipeptidase fromEscherichia coli†,‡." Biochemistry 42.17 (2003): 4874-882. Web.
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The active inhibitory site containing the scissile bond is located in the loop between beta-strands 4 and 5 in STI and ETI. The STIs belong to a superfamily that also contains the interleukin-1 proteins, heparin binding growth factors (HBGF) and histactophilin, all of which have very similar structures, but share no sequence similarity with the STI family.
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The crystal structure of a four-way junctional twister sister ribozyme is different from the three-way junctional one in terms of long-range interaction and active site structure. The active site of a four-way junctional twister sister is splayed-apart with an interaction between guanine and scissile phosphate. Besides, there are seven divalent metal ions in this ribozyme.
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The divalent metal ions identified in the tertiary structure are shown as green balls. d Highly conserved residues (shown in red) are brought into close proximity by the interaction between partially zippered-up L1 and SL4 loops in the tertiary fold of the twister-sister ribozyme The crystal structures of the pre-catalytic state of the twister sister ribozymes were solved by two research groups independently. The structure of a three-way junctional twister sister ribozyme is composed of two co-axial stacked helical sections connected with a three-way junction and two tertiary contacts. The active site, a scissile phosphate, is located in a loop with quasihelical character in one coaxial base-stacked helix. Five divalent metal ions are coordinate to RNA ligands, one of which is directly bound to C54 O2’ near the scissile phosphate and exchange inner sphere water molecules with the RNA ligands.
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In vitro, DiGIR1 catalyses three different reactions. The first one consists in hydrolysis of the scissile phosphate at the IPS site. This is the cleavage reaction observed with the full-length intron and several length variants with a relative low rate. The hydrolytic cleavage is irreversible and is considered an in vitro artefact resulting from misfolding of the catalytic site to present the branch nucleotide (BP) correctly for the reaction.
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In her study, published in September 2016, Hammes-Schiffer contributed towards discovering the effects of the active site of the magnesium ion in the Scissile Phosphate cofactor complex. She discovered that rather than the magnesium ion lying in the center of the complex, the ion lies in a separate site, termed the Hoogsteen Face, where it lowers the pKa of the complex in order to facilitate a deprotonation reaction necessary for a self-cleavage reaction.
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Replacements of the pro-S nonbridging oxygen of the scissile phosphate with a thiol group leads to reduced self-cleavage rates, suggesting that the mechanism is not reliant on bound magnesium. Rescue of the thiol- derivative by cadmium cations indicates that divalent metal ions play a role in rate enhancement. A likely mechanism for this is the stabilization of the transition state by reducing electrostatic strain on the substrate strand from the growing negative charge during cleavage.
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10 nucleotides were discovered to be highly conserved amongst many pistol ribozymes: G5, A19, A20, A 21, A31, A32, A33, G40, C41, and G42. Mutation to any of these nucleotides disrupt its secondary structure, which also disrupt its catalytic ability. The scissile bond was also determine to be between G53-U54 located in the junction connecting P2 and P3. Although the identity of these two nucleotides might vary, the length of the junction remains highly conserved.
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It was however completely unclear how G12 and G8 could accomplish this, given the original structures of the minimal hammerhead ribozyme. Other concerns included an NOE between U4 and U7 of the cleaved hammerhead ribozyme that had also been observed during NMR characterization, which suggested that these nucleotide bases must approach one another closer than about 6 Å, although close approach of U7 to U4 did not appear to be possible from the crystal structure. Finally, as previously discussed, the attacking nucleophile in the original structures, the 2’-OH of C17, was not in a position amenable to in-line attack upon the adjacent scissile phosphate. Perhaps most worrisome were experiments that suggested the A-9 and scissile phosphates must come within about 4 Å of one another in the transition-state, based upon double phosphorothioate substitution and soft metal ion rescue experiments; the distance between these phosphates in the minimal hammerhead crystal structure was about 18 Å, with no clear mechanism for close approach if the Stem II and Stem I A-form helices were treated as rigid bodies.
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The hydrolysis of ADPR is catalyzed by E162, which improves the nucleophilicity of the water molecule in the active site by deprotonating it. This water is held perfectly in line with the scissile bond by the first and second magnesium ions. The hydroxide ion then attacks the phosphorus atom on the adenosyl phosphate, creating a trigonal bypyramidal intermediate with a negatively charged oxygen attached to the adenosyl phosphate. The double bond is then reformed, effectively discharging ribose 5-phosphate as a leaving group.
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This forms a tetrahedral gem- diolate intermediate that is coordinated to the zinc atom. In order for the amide product to be released from the active site, the scissile amide must abstract a second proton from the coordinated water molecule. Alternatively, it has been shown for thermolysin (another metalloproteinase) that the amide product can be released in its neutral (R-NH2) form. The carboxylate product is released after a water molecule attacks the zinc ion and displaces the carboxylate product.
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The indole nitrogen of tryptophan 315 also forms a hydrogen bond to this scissile phosphate. (n.b A Histidine occupies this site in other tyrosine recombinase family members and performs the same function). This reaction cleaves the DNA and frees a 5’ hydroxyl group. This process occurs in the active site of two out of the four recombinase subunits present at the synapse tetramer. If the 5’ hydroxyl groups attack the 3’-phosphotyrosine linkage one pair of the DNA strands will exchange to form a Holliday junction intermediate.
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This is probably the source of the observation that divalent metal ions are required at low ionic strength, but can be dispensed with at higher concentrations of monovalent cations. The reaction thus likely involves abstraction of the 2'-proton from C17, followed by nucleophilic attack upon the adjacent phosphate. As the bond between the scissile phosphorus and the 5'-O leaving group begins to break, a proton is supplied from the ribose of G8, which then likely reprotonates at the expense of a water molecule observed to hydrogen bond to it in the crystal structure.
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If the invariant G8 is changed to C8, hammerhead catalysis is abolished. However, a G8C + C3G double-mutant that maintains the G8-C3 base pair found in the full-length hammerhead restores most of the catalytic activity. The 2'-OH of G8 has also been observed to be essential for catalysis; replacement of G8 with deoxyG8 greatly reduces the rate of catalysis, suggesting the 2'-OH is indeed crucial to the catalytic mechanism. The close approach of the A9 and scissile phosphates requires the presence of a high concentration of positive charge.
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AMPCPR is a nonhydrolyzable analog of ADP-ribose, and thus functions as an inhibitor of ADPRase. The picture below reveals the first and second Mg2+ ions, which serve to coordinate the attacking water molecule so that it is perfectly in line with the scissile bond (O-P-O bond is 177 degrees) and poised for attack. A glutamate side chain (E162) will deprotonate the water molecule, and then the hydroxide ion will attack nucleophilically. This occurs when the water molecule is 3.0 angstroms away from the phosphorus molecule it is attacking.
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And it was assumed that both their mechanism of action and their active site might be similar. A positively charged Arg145 at the active site was thought to bind with the negatively charged C-terminal carboxyl group of the peptide substrate. It was also proposed that ACE binds by hydrogen bonding to the terminal, non scissile, peptide bond of the substrate. But since ACE is a dipeptide carboxypeptidase, unlike carboxypeptidase A, the distance between the cationic carboxyl-binding site and the zinc atom should be greater, by approximately the length of one amino acid residue.
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Kex2 was first purified and characterized by Charles Brenner and Robert Fuller in 1992. The Kex2 crystal structure was solved by a group led by Dagmar Ringe, Robert Fuller and Gregory Petsko. That of Furin was determined by a group led by Manual Than and Wolfram Bode. The key features of Kex2 and Furin are a subtilisin-related catalytic domain, a specificity pocket that requires the amino acid amino terminal to the scissile bond to be arginine for rapid acylation, and a P-domain carboxy-terminal to the subtilisin domain, which is required for biosynthesis.
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The mechanism for prolidase catalytic activity remains largely uncharacterized. However, biochemical and structural analyses of aminopeptidase (APPro), methionine aminopeptidase (MetAP), and prolidase, all members of the “pita-bread” metalloenzymes, suggest that they share a common mechanism scheme. The main difference arises in the location of the carbonyl oxygen atom of the scissile peptide bond. Proposed mechanism scheme for metal- dependent "pita-bread" enzyme with eMetAP residue numbering. The following mechanism shows a proposed scheme for a metal-dependent “pita-bread” enzyme with residue numbering corresponding to those found in methionine aminopeptidase from E. coli.
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HUH endonucleases generally have two histidine (H) residues in the active site coordinating a metal cation (Mg2+ or Mn2+) that interacts with the phosphate backbone of DNA. This allows for a nucleophilic attack, most commonly, by an activated tyrosine of the scissile phosphate in the DNA backbone, generating a 5' covalent bond with the ssDNA. In contrast to other DNA-protein linkage approaches, this reaction occurs at ambient conditions and does not require any additional modifications. X-ray crystallography and NMR structures have provided insight into the sequence specificity of DNA binding.
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Three-dimensional structure of the full-length hammerhead ribozyme In 2006 a 2.2 Å resolution crystal structure of the full-length hammerhead ribozyme was obtained. This new structure (shown on the right) appears to resolve the most worrisome of the previous discrepancies. In particular, C17 is now positioned for in-line attack, and the invariant residues C3, G5, G8 and G12 all appear involved in vital interactions relevant to catalysis. Moreover, the A9 and scissile phosphates are observed to be 4.3 Å apart, consistent with the idea that, when modified, these phosphates could bind a single thiophilic metal ion.
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The active site of OmpT resembles that of other omptins, and is characterized by conserved residues at Asp84, Asp86, Asp206, and His208. The most common bond cleavage by OmpT is between two arginine residues because their positive charge can favorably interact with the negatively charged species at the active site during substrate binding. Because of the specificity of the active site, OmpT does not act on peptides with a negatively charged residue adjacent to the scissile bond. Also, OmpT is specifically identified an endopeptidase because it does not cleave peptides at the N- or C-terminus, but only between nonterminal amino acids.
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Consequently, when two Cre molecules bind at a single loxP site two active sites are present. Cre mediated recombination requires the formation of a synapse in which two Cre-LoxP complexes associate to form what is known as the synapse tetramer in which 4 distinct active sites are present. Tyr 324 acts as a nucleophile to form a covalent 3’-phosphotyrosine linkage to the DNA substrate. The scissile phosphate (phosphate targeted for nucleophilic attack at the cleavage site) is coordinated by the side chains of the 3 amino acid residues of the catalytic triad (Arg 173, His 289 & Trp 315).
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Chymotrypsin (, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the S1 position) of the enzyme. It is activated in the presence of trypsin.
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The Mg2+ ion on the right (Figure 3) interacts with negatively charged oxygens of the alpha(α), beta(β) and gamma(γ) phosphates to align the scissile bond for the primer to attack. Even if there is no general base within the active site to deprotonate the primer hydroxyl, the lowered pka of the metal-bound hydroxyl favors the formation of the 3’-hydroxide nucleophile. Metal ions and Lys522 contact non- bridging oxygens on the α-phosphate to stabilize the negative charge developing on the α-phosphorus during bond formation with the nucleophile. Moreover, the Lys522 sidechain also moves to neutralize the negatively charged pyrophosphate group.
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The reaction is therefore reversible, as the scissile phosphate remains a phosphodiester, and may thus act as a substrate for hammerhead RNA- mediated ligation without a requirement for ATP or a similar exogenous energy source. The hammerhead ribozyme-catalyzed reaction, unlike the formally identical non-enzymatic alkaline cleavage of RNA, is a highly sequence- specific cleavage reaction with a typical turnover rate of approximately 1 molecule of substrate per molecule of enzyme per minute at pH 7.5 in 10 mM Mg2+ (so-called “standard reaction conditions” for the minimal hammerhead RNA sequence), depending upon the sequence of the particular hammerhead ribozyme construct measured. This represents an approximately 10,000-fold rate enhancement over the nonezymatic cleavage of RNA.
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The alpha-macroglobulin (aM) family of proteins includes protease inhibitors, typified by the human tetrameric alpha-2-macroglobulin (a2M); they belong to the MEROPS proteinase inhibitor family I39, clan IL. These protease inhibitors share several defining properties, which include (i) the ability to inhibit proteases from all catalytic classes, (ii) the presence of a 'bait region' (aka. a sequence of amino acids in an α2-macroglobulin molecule, or a homologous protein, that contains scissile peptide bonds for those proteinases that it inhibits) and a thiol ester, (iii) a similar protease inhibitory mechanism and (iv) the inactivation of the inhibitory capacity by reaction of the thiol ester with small primary amines. aM protease inhibitors inhibit by steric hindrance. The mechanism involves protease cleavage of the bait region, a segment of the aM that is particularly susceptible to proteolytic cleavage, which initiates a conformational change such that the aM collapses about the protease.
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The C75 in the HDV ribozyme has been the subject of several studies because of its peculiar pKa. The typical pKa values for the free nucleosides are around 3.5 to 4.2; these lower pKa values are acidic and it is unlikely that they would become basic. However, it is likely that the structural environment within the ribozyme, which includes a desolvated active site cleft, provides negative electrostatic potential that could perturb the pKa of cytosine enough to act as a Lewis acid.General acid catalysis by cytosine 75, in which the protonated form of the C donates a proton to the leaving group during catalysis In addition to Lewis acid stabilization of the 5′-hydroxyl leaving group, it is also now accepted that the HDV ribozyme can use a metal ion to assist in activation of the 2′-hydroxyl for attack on the U(-1) nucleotide. A magnesium ion in the active site of the ribozyme is coordinated to the 2’-hydroxyl nucleophile and an oxygen of the scissile phosphate, and may act as a Lewis acid to activate the 2′-hydroxyl.
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