Sandbox Reserved 994: Difference between revisions

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Due to overperscription and misuse of antibiotics, bacteria have been able to develop resistance mechanisms. One of these resistance mechanisms is through the expression of β-lactamases, which have evolved as a seperate enzyme over millions of years from PBP.<ref>Meroueh, S.O; Minasov, G; Lee, W; Shoichet, B.K; Mobashery, S. Structural aspects for evolution of beta-lactamases from penicillin-binding proteins. J. Am. Chem Soc. (2003), 125, 9612-9618. </ref> β-lactamases act by hydrolyzing the β-lactam ring, which renders the antibiotic inactive before it has a chance to inhibit the transpeptidase enzymes.<ref>Neu, Harold. "The Crisis in Antibiotic Resistance." Science (1992) 257, 5073. ProQuest Medical Library: p. 1064-1072.</ref> β-lactamases are grouped into four different classes (A, B, C and D), which all (besides class B) use a serine based mechanism for destruction of β-lactams. Class B β-lactamases use zinc ions for hydrolysis. Class D was distinguished from other serine β-lactamases in the late 1980s, due to having an affinity for oxacillin as its substrate in addition to other antibiotics.<ref>doi: 10.1128/AAC.01009-09</ref> Even more concerning is that the class D β-lactamases, or OXAs, are not inhibited by current clinical β-lactamase inhibitors, such as clavulanic acid. OXA-24, which has considerable carbapenemase activity, poses a dangerous clinical threat due to the absence of an effective inhibitor.

~~== CHDLs ==~~

== Structure ==

The active site of OXA-24 is characterized by a hydrophobic pocket, which is representative of Class D β-lactamases as a whole, and a ~~disulfide bridge~~. The mechanism of attack is through the use of three catalytic residues: Serine-81, Carboxylated Lysine-84, and Serine-~~115~~.

OXA-24 is a monomeric protein. The active site is composed of a short <nowiki>310</nowiki> helix and a β-sheet.<ref>DOI: 10.1021/ar300327a</ref> The active site of OXA-24 is characterized by a hydrophobic pocket, which is representative of Class D β-lactamases as a whole. The hydrophobic bridge contributes to the substrate specificity for carbapenems and is composed of an arrangement of the Tyr-112 and Met-223 side chains. <ref>doi:10.1073/pnas.0607557104</ref> These residues block the active site and only allow a very specific binding configuration of antibiotics. The active site is overall positively charged and contains a sulfate ion along with other solvent molecules when no substrate is bound. The mechanism of attack is through the use of three catalytic residues: Serine-81, Carboxylated Lysine-84, and Serine-128. The hydroxyl chain of Ser-128 conforms in the direction of the active-serine Ser-81, and contributes to the catalytic mechanism.<ref>doi:10.1073/pnas.0607557104</ref>

== Hydrolysis Mechanism ==

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<scene name='69/691536/Closeupdrug/1'>close up</scene><ref>PMID: 10817708</ref>

~~== Mechanistic Activity ==~~

There are three catalytic residues involved in the hydrolysis of β-lactam antibiotics. Serine-81 (S81) is the catalytic serine, which preforms a nucleophilic attack on the β-lactam ring after being deprotonated by the carboxylated lysine-84 (KCX84). This carboxylated lysine is formed by CO2 in the environment engaging in unfavorable interactions with the hydrophobic pocket of OXA-24, so it carboxylates Lysine-84.

The second step forms a high energy intermediate~~, where protons are shifted around the newly-formed complex~~. The cyclic amide from the β-lactam deprotonates Serine-128 (S128), which proceeds to deprotonate the amine on KCX84, which deprotonates the carboxylate group. This high energy intermediate resolves to form the stable acyl-enzyme intermediate.

There are three catalytic residues involved in the hydrolysis of β-lactam antibiotics. Serine-81 is the catalytic serine, which performs a nucleophilic attack on the β-lactam ring after being deprotonated by the carboxylated lysine-84 (KCX84). This carboxylated lysine is formed by CO2 in the environment engaging in unfavorable interactions with the hydrophobic pocket of OXA-24, so it carboxylates Lysine-84 (A).

The second step (B) forms a high energy intermediate. The cyclic amide from the β-lactam deprotonates Serine-128 (S128), which proceeds to deprotonate the amine on KCX84, which deprotonates the carboxylate group. This high energy intermediate resolves to form the stable acyl-enzyme intermediate.

The third step proceeds with the use of a catalytic water, which is deprotonated by KCX84. The water now can mount a nucleophilic attack on the ester linkage connecting S81 and the hydrolyzed β-lactam. This forms a high energy dissociation intermediate, where S81 is released by a mechanism, which has not quite yet been determined. It is suspected that it deprotonates KCX84, but this has not yet been confirmed.

The third step (C) proceeds with the use of a catalytic water, which is deprotonated by KCX84. The water now can mount a nucleophilic attack on the ester linkage connecting S81 and the hydrolyzed β-lactam. This forms a high energy dissociation intermediate, where S81 is released by a mechanism, which has not quite yet been determined. It is suspected that it deprotonates KCX84, but this has not yet been confirmed.

In step 4, The enzyme is successfully regenerated and the hydrolyzed β-lactam antibiotic is released back into solution. OXA-24 is now free to hydrolyze another substrate and the antibiotic has been rendered useless.

In step four (D), The enzyme is successfully regenerated and the hydrolyzed β-lactam antibiotic is released back into solution. OXA-24 is now free to hydrolyze another substrate and the antibiotic has been rendered useless.

This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.

Sandbox Reserved 994: Difference between revisions

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@@ Line 13: / Line 13: @@
 Due to overperscription and misuse of antibiotics, bacteria have been able to develop resistance mechanisms. One of these resistance mechanisms is through the expression of β-lactamases, which have evolved as a seperate enzyme over millions of years from PBP.<ref>Meroueh, S.O; Minasov, G; Lee, W; Shoichet, B.K; Mobashery, S. Structural aspects for evolution of beta-lactamases from penicillin-binding proteins. J. Am. Chem Soc. (2003), 125, 9612-9618. </ref> β-lactamases act by hydrolyzing the β-lactam ring, which renders the antibiotic inactive before it has a chance to inhibit the transpeptidase enzymes.<ref>Neu, Harold. "The Crisis in Antibiotic Resistance." Science (1992) 257, 5073. ProQuest Medical Library: p. 1064-1072.</ref> β-lactamases are grouped into four different classes (A, B, C and D), which all (besides class B) use a serine based mechanism for destruction of β-lactams. Class B β-lactamases use zinc ions for hydrolysis. Class D was distinguished from other serine β-lactamases in the late 1980s, due to having an affinity for oxacillin as its substrate in addition to other antibiotics.<ref>doi:  10.1128/AAC.01009-09</ref> Even more concerning is that the class D β-lactamases, or OXAs, are not inhibited by current clinical β-lactamase inhibitors, such as clavulanic acid. OXA-24, which has considerable carbapenemase activity, poses a dangerous clinical threat due to the absence of an effective inhibitor.
-== CHDLs ==
 == Structure ==
-The active site of OXA-24 is characterized by a hydrophobic pocket, which is representative of Class D β-lactamases as a whole, and a disulfide bridge. The mechanism of attack is through the use of three catalytic residues: Serine-81, Carboxylated Lysine-84, and Serine-115.
+OXA-24 is a monomeric protein. The active site is composed of a short <nowiki>310</nowiki> helix and a β-sheet.<ref>DOI: 10.1021/ar300327a</ref> The active site of OXA-24 is characterized by a hydrophobic pocket, which is representative of Class D β-lactamases as a whole. The hydrophobic bridge contributes to the substrate specificity for carbapenems and is composed of an arrangement of the Tyr-112 and Met-223 side chains. <ref>doi:10.1073/pnas.0607557104</ref> These residues block the active site and only allow a very specific binding configuration of antibiotics. The active site is overall positively charged and contains a sulfate ion along with other solvent molecules when no substrate is bound. The mechanism of attack is through the use of three catalytic residues: Serine-81, Carboxylated Lysine-84, and Serine-128. The hydroxyl chain of Ser-128 conforms in the direction of the active-serine Ser-81, and contributes to the catalytic mechanism.<ref>doi:10.1073/pnas.0607557104</ref>
 == Hydrolysis Mechanism ==
@@ Line 27: / Line 25: @@
 <scene name='69/691536/Closeupdrug/1'>close up</scene><ref>PMID: 10817708</ref>
-== Mechanistic Activity ==
-There are three catalytic residues involved in the hydrolysis of β-lactam antibiotics. Serine-81 (S81) is the catalytic serine, which preforms a nucleophilic attack on the β-lactam ring after being deprotonated by the carboxylated lysine-84 (KCX84). This carboxylated lysine is formed by CO2 in the environment engaging in unfavorable interactions with the hydrophobic pocket of OXA-24, so it carboxylates Lysine-84.
-The second step forms a high energy intermediate, where protons are shifted around the newly-formed complex.  The cyclic amide from the β-lactam deprotonates Serine-128 (S128), which proceeds to deprotonate the amine on KCX84, which deprotonates the carboxylate group. This high energy intermediate resolves to form the stable acyl-enzyme intermediate.
+There are three catalytic residues involved in the hydrolysis of β-lactam antibiotics. Serine-81 is the catalytic serine, which performs a nucleophilic attack on the β-lactam ring after being deprotonated by the carboxylated lysine-84 (KCX84). This carboxylated lysine is formed by CO2 in the environment engaging in unfavorable interactions with the hydrophobic pocket of OXA-24, so it carboxylates Lysine-84 (A).
+The second step (B) forms a high energy intermediate. The cyclic amide from the β-lactam deprotonates Serine-128 (S128), which proceeds to deprotonate the amine on KCX84, which deprotonates the carboxylate group. This high energy intermediate resolves to form the stable acyl-enzyme intermediate.
-The third step proceeds with the use of a catalytic water, which is deprotonated by KCX84. The water now can mount a nucleophilic attack on the ester linkage connecting S81 and the hydrolyzed β-lactam. This forms a high energy dissociation intermediate, where S81 is released by a mechanism, which has not quite yet been determined. It is suspected that it deprotonates KCX84, but this has not yet been confirmed.
+The third step (C) proceeds with the use of a catalytic water, which is deprotonated by KCX84. The water now can mount a nucleophilic attack on the ester linkage connecting S81 and the hydrolyzed β-lactam. This forms a high energy dissociation intermediate, where S81 is released by a mechanism, which has not quite yet been determined. It is suspected that it deprotonates KCX84, but this has not yet been confirmed.
-In step 4, The enzyme is successfully regenerated and the hydrolyzed β-lactam antibiotic is released back into solution. OXA-24 is now free to hydrolyze another substrate and the antibiotic has been rendered useless.
+In step four (D), The enzyme is successfully regenerated and the hydrolyzed β-lactam antibiotic is released back into solution. OXA-24 is now free to hydrolyze another substrate and the antibiotic has been rendered useless.
 This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes.