Ketosteroid Isomerase: Difference between revisions
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===Hydrophobic Active Site=== | ===Hydrophobic Active Site=== | ||
[[Image:phobia.png|left]] | [[Image:phobia.png|thumb|left|'''Hydrophobic active site with bound equilenin.''']] | ||
KSI's active site is located within a hydrophobic cavity formed helices B and C crossing over the "front face" of the β-sheet that is approximately 8.5 by 9.5 Å at its opening and is 16 Å deep.<ref name="Wu" /> The cavity is lined with <scene name='User:Laura_M._Haynes/Sandbox_1/Beta-phobics/1'>hydrophobic "front facing" residues</scene> from the β-sheet(Val36, Pro39, Leu63, Val65, Leu67, Val71, Phe80, Phe82, Val84, Val95, Pro97, Phe101, Ala114, and Phe116). <scene name='User:Laura_M._Haynes/Sandbox_1/Helix-phobic/1'>Hydrophobic residues on the other side of the cavity</scene> contributed from the α-helices include: Val11, Tyr14, Val15, Leu18, Phe54, and Tyr55.<ref name="Wu" /> | KSI's active site is located within a hydrophobic cavity formed helices B and C crossing over the "front face" of the β-sheet that is approximately 8.5 by 9.5 Å at its opening and is 16 Å deep.<ref name="Wu" /> The cavity is lined with <scene name='User:Laura_M._Haynes/Sandbox_1/Beta-phobics/1'>hydrophobic "front facing" residues</scene> from the β-sheet(Val36, Pro39, Leu63, Val65, Leu67, Val71, Phe80, Phe82, Val84, Val95, Pro97, Phe101, Ala114, and Phe116). <scene name='User:Laura_M._Haynes/Sandbox_1/Helix-phobic/1'>Hydrophobic residues on the other side of the cavity</scene> contributed from the α-helices include: Val11, Tyr14, Val15, Leu18, Phe54, and Tyr55.<ref name="Wu" /> | ||
Although the <scene name='User:Laura_M._Haynes/Sandbox_1/Active_site_1isk/1'>active site</scene> of KSI is notably hydrophobic, it contains several hydrophilic residues believed to be important to the enzymatic function of the protein. The hydrophobic active site of KSI contains an aspartate residue at position 99 and a tyrosine residue at position 14 (according to the numbering for the ''Commamonas tetosteroni'' protein, which will be used throughout) that are capable of forming hydrogen bonds with the 3-position carbonyl of the steroid and form an active site oxyanion hole.<ref name="Pollack" />,<ref name="Sigala2008">PMID:18808119 </ref> Additionally, the active site contains an aspartate residue at position 38 that is participates in the catalytic activity of KSI.<ref name="Pollack" /> [[Image:cis.png|right]] | Although the <scene name='User:Laura_M._Haynes/Sandbox_1/Active_site_1isk/1'>active site</scene> of KSI is notably hydrophobic, it contains several hydrophilic residues believed to be important to the enzymatic function of the protein. The hydrophobic active site of KSI contains an aspartate residue at position 99 and a tyrosine residue at position 14 (according to the numbering for the ''Commamonas tetosteroni'' protein, which will be used throughout) that are capable of forming hydrogen bonds with the 3-position carbonyl of the steroid and form an active site oxyanion hole.<ref name="Pollack" />,<ref name="Sigala2008">PMID:18808119 </ref> Additionally, the active site contains an aspartate residue at position 38 that is participates in the catalytic activity of KSI.<ref name="Pollack" /> [[Image:cis.png|thumb|right|'''Cis-Pro39.''']] | ||
Upon substrate binding the the three α-helices become more tightly packed with the "front face" of the β-sheet. This in turn allows Tyr14 to approach Asp99 and the substrate. | Upon substrate binding the the three α-helices become more tightly packed with the "front face" of the β-sheet. This in turn allows Tyr14 to approach Asp99 and the substrate. | ||
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Pro39 of KSI participates in a cis-peptide linkage with Asp38 in forming a <scene name='User:Laura_M._Haynes/Sandbox_1/Cis_loop/1'>four-residue linkage</scene>between stands 1 and 2 of the 6 stranded beta-sheet.<ref name="Wu" /> The cis-peptide linkage serves to correctly position the key catalytic residue Asp38 within the active site. Mutating residue to Gly or Ala results in the improper positioning of Asp38 within the active site leading to corresponding 2-fold decreases in enzyme's catalytic efficiency.<ref name="NAM">PMID:12852789 </ref> The cis-peptide bond also helps to stablize and increase the rigidity of the four-residue linkage between β-strands 1 and 2.<ref name="NAM" /> | Pro39 of KSI participates in a cis-peptide linkage with Asp38 in forming a <scene name='User:Laura_M._Haynes/Sandbox_1/Cis_loop/1'>four-residue linkage</scene>between stands 1 and 2 of the 6 stranded beta-sheet.<ref name="Wu" /> The cis-peptide linkage serves to correctly position the key catalytic residue Asp38 within the active site. Mutating residue to Gly or Ala results in the improper positioning of Asp38 within the active site leading to corresponding 2-fold decreases in enzyme's catalytic efficiency.<ref name="NAM">PMID:12852789 </ref> The cis-peptide bond also helps to stablize and increase the rigidity of the four-residue linkage between β-strands 1 and 2.<ref name="NAM" /> | ||
== | ==Protein Folding== | ||
The work of Kim et al. suggests that KSI assembles into its biologically active dimer structure via a multistep pathway.<ref name="folding" /> The monomers initially exist in an unfolded configuration (U) with Pro39 in a trans configuration. The monomers then fold into an intermediate configuration that is capable of binding transition state analogs (I). At this stage the native secondary structures have most likely formed, however, it is also likely that hydrophobic residues have been internalized into the core. The tertiary structure formation is most likely promoted by the dimerized intermediate structure (I') before folding to the final native structure. Cis-trans isomerization is the rate-limiting step with dimer structures being formed preferentially from cis-Pro39 monomers. | |||
[[Image:folding.png|thumb|center|'''Protein folding pathway of KSI.''' (U) Unfolded protein. (I) Monomer intermediate. (I') Dimerized intermediate.]] | |||
==Enzymology== | |||
<applet load='1QJG' size='300' frame='true' align='left' caption='A monomer of ketosteroid isomerase is shown in complex with the transition state analog [http://en.wikipedia.org/wiki/Equilenin equinelin] (PBD Structure ID [[1qjg|1QJG]]).' scene='User:Laura_M._Haynes/Sandbox_1/1qjg/1'/> | <applet load='1QJG' size='300' frame='true' align='left' caption='A monomer of ketosteroid isomerase is shown in complex with the transition state analog [http://en.wikipedia.org/wiki/Equilenin equinelin] (PBD Structure ID [[1qjg|1QJG]]).' scene='User:Laura_M._Haynes/Sandbox_1/1qjg/1'/> | ||
===General Mechanism=== | ===General Mechanism=== | ||
