Allen sandbox 1: Difference between revisions

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Justin Allen

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 {{STRUCTURE_1one |  PDB=1one  |  SCENE=  }}
-<scene name='Cory_Tiedeman_Sandbox_1/Enolase/1'>Enolase</scene> is an enzyme that catalyzes the reversible dehydration reaction of 2-phosphoglycerate (2PG) into Phosphophenolglycerate (PEP) in the 9th reactiion of glycolysis.<ref>{{textbook |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=500|}}</ref>.  Glycolysis converts glucose into two 3-carbon molecules called pyruvate.  The energy released during glycolysis is used to make ATP.<ref>{{textbook |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=487|}}</ref>.  Enolase is expressed abundantly in most cells and has been proven useful as a model to study mechanisms of enzyme action and structural analysis, especially for those enzymes involved in glycolysis <ref>{{journal}}</ref>.
+<scene name='Cory_Tiedeman_Sandbox_1/Enolase/1'>Enolase</scene> is an enzyme that catalyzes the reversible dehydration reaction of 2-phosphoglycerate (2PG) into phosphophenolglycerate (PEP) in the 9th reaction of glycolysis.<ref>{{textbook |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=500|}}</ref>  Glycolysis converts glucose into two 3-carbon molecules called pyruvate.  The energy released during glycolysis is used to make ATP.<ref>{{textbook |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=487|}}</ref>  Enolase is expressed abundantly in most cells and has been proven useful as a model to study mechanisms of enzyme action and structural analysis, especially for those enzymes involved in glycolysis <ref>{{journal}}</ref>.
 ==Structure==
-The <scene name='Cory_Tiedeman_Sandbox_1/Secondary_structure/1'>secondary structure</scene> of enolase contains both alpha helices and beta sheets.  The beta sheets are mainly parallel<ref>{{web site| title=SCOP: Protein: Enolase from Baker's yeast (Saccharomyces cerevisiae)|url=http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.d.b.bc.b.b.html|}}</ref>.  As shown in the figure, enolase has about 36 alpha helices and 22 beta sheets (18 alpha helices and 11 beta sheets per domain).  Enolase consists of two domains.
+[[Image:Enolase_with_differentiated_subunits.jpg|300px|left|thumb| Tram Nguyen & Katelyn Thompson; Differentiated subunits, in parallel orientation, of enolase using UCSF Chimera (PDB 1ONE)]]
+The <scene name='Cory_Tiedeman_Sandbox_1/Secondary_structure/1'>secondary structure</scene> of enolase contains both alpha helices and beta sheets.  The beta sheets are mainly parallel<ref>{{web site| title=SCOP: Protein: Enolase from Baker's yeast (Saccharomyces cerevisiae)|url=http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.d.b.bc.b.b.html|}}</ref>.  As shown in the figure, enolase has about 36 alpha helices and 22 beta sheets (18 alpha helices and 11 beta sheets per domain), resulting in a molecular weight of 82,000-100,000 Daltons.  Enolase consists of two domains paired together in antiparallel orientation.
 '''Structural Clasification of Proteins (SCOP)<ref>{{web site| title=SCOP: Protein: Enolase from Baker's yeast (Saccharomyces cerevisiae)|url=http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.d.b.bc.b.b.html|}}</ref>'''
-Enolase is in the alpha and beta proteins class and has a fold of TIM beta/alpha-barrel.  It comes from the Superfamily on Enolase C-terminal domain-like and is in the enolase family.
+Enolase is in the Alpha and Beta protein class (mainly parallel beta sheets) and has a fold of TIM beta/alpha-barrel, which consists of parallel barrels composed of beta sheets.  Enolase also belongs to the Enolase C-terminal domain-like protein Superfamily, which means it can bind metal ions (magnesium or manganese) in a conserved site inside barrel N-terminal alpha+beta domains.
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 [[Image:mechanism.png|left|400px|The mechanism of 2PG to PEP using enolase.]]<ref>{{website2}}</ref>
 The
-<scene name='Cory_Tiedeman_Sandbox_1/Active_site/1'>active site</scene> of enolase as shown, involves Lys 345, Lys 396, Glu 168, Glu 211, and His 159.  Enolase forms a complex with two
+<scene name='Cory_Tiedeman_Sandbox_1/Active_site/1'>active site</scene> of enolase includes the residues Lys 345, Lys 396, Glu 168, Glu 211, and His 159.  Enolase forms a complex with two
 <scene name='Cory_Tiedeman_Sandbox_1/Mg/3'>Mg 2+'s</scene> at its active site.
-The substrate, 2PG, binds to the two <scene name='Cory_Tiedeman_Sandbox_1/Mechanism/4'>Mg2+'s, Glu 211, and Lys 345</scene>.  The Mg 2+ then forms a bond at the deprotonated carboxylic acid on the 1'C to connect it with enolase.  It also is connects to Glu 211 and Lys 345.  Glu 211 makes a hydrogen bond with the alcohol group on the 3'C.  Lys 345 deprotonates the 2'C and then the 2'C forms an alkene with the 1'C which then moves the electrons forming the ketone onto the oxygen making it have a negative charge.  The other oxygen, which already has a negative charge, then moves its electron to form a ketone with the 1'C.  The electrons that made up the alkene between the 1'C adn 2'C then moves to form an alkene between the 2'C and 3'C.  This breaks the bond with the alcohol on the 3'C which deprotonates Glu 211 on enolase to form H2O.  Then the new molecule is released from enolase as PEP.  PEP then goes on through another step in glycolysis to create pyruvate.
+The mechanism of enolase follows 3 steps:
+'''Step 1''': The substrate, 2PG, binds to the two <scene name='Cory_Tiedeman_Sandbox_1/Mechanism/4'>Mg2+'s</scene>. The carboxyl group coordinates with the two magnesium ions, which stabilizes the negative charge on the oxygen atom and removes charge from the alpha hydrogen, making it a better leaving group.
+'''Step 2''': Lys 345 then deprotonates the alpha hydrogen, a reaction which is stabilized by resonance between the carboxyl oxygens and the two Mg ions and Glu 211 bonded to the hydroxyl group. This creates a carbanion intermediate.
+'''Step 3''': An electron transfer reaction then occurs from the C'1 carboxyl oxygen to form a ketone. This removes electrons from the alkene bond between C'1 and C'2 to create an alkene between C'2 and C'3 instead. This allows the C'3 hydroxyl group to deprotonate Glu 211, resulting in the ejection of a water molecule and formation of the product PEP. PEP is then dephosphorylated in the next step of glycolysis to create pyruvate.
+Studies have shown that mutating the residues within the active site can have serious effects on the activity of the enzyme. For example, mutation in either Glu 168, Glu 211, Lys 345, or Lys 396 of the active site results in a reduction in the reaction activity by 10^4 to 10^5 of wild type levels. Clearly, these residues play critical roles in maintaining enolase's activity.<ref>Reed, G., Poyner, R., Larsen, T., Wedekind, J., Rayment, I. (1996)''Current Opinion in Structural Biology'' '''6''':736-743</ref>
+Additionally, this mechanism can be inhibited by addition of Fluoride ions, which bond to Mg 2+, thus blocking the substrate (2PG) from binding to the active site of enolase.<ref>{{textbook |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=500|}}</ref>
-Fluoride ions inhibits glycolysis by forming a bond with Mg 2+ thus blocks the substrate (2PG) from binding to the active site of enolase.<ref>{{textbook |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=500|}}</ref>
 ==Kinetics==
-[[Image:enolase kinetics.jpeg|left|150px|V vs. [PGA]; PGA is 2PG, the top curve has [Mg2+] of 10^-3 M and the bottom curve has [Mg2+] of 106-2 M]]<ref>{{journal2}}</ref>
+[[Image:enolase kinetics.jpeg|left|150px|V vs. [PGA]; PGA is 2PG, the top curve has [Mg2+] of 10^-3 M and the bottom curve has [Mg2+] of 106-2 M]]<ref>{{journal2}}</ref>The kinetics of the enolase reaction can be affected by the concentration of magnesium ion, Mg2+. The graph to the left shows velocity vs. [PGA], in which PGA stands for 2-PG. The upper curve is the reaction at normal [Mg2+], 0.01 M, while the lower curve shows the same reaction at an increased concentration of [Mg2+], 0.1 M. This shows that upon addition of Mg2+, the Vmax is lowered to sub-optimal levels, while the Km remains relatively unchanged. Therefore, the upper curve (lower [Mg2+]) is more desirable because it achieves a greater Vmax at essentially the same Km value; a greater Vmax can be attained without the need for additional substrate<ref>{{journal2}}</ref>.
-Since Mg2+ is essential for binding the substrate, 2-PG, it is also needed at a specific quality in order to have a good rate, or velocity.  The graph shows the V vs. [PGA], in which PGA is 2-PG, with two different concentrations of Mg2+.  The upper curve, which also has greater Vmax, has an Mg2+ concentration of 10^-3 M while the lower curve, which has a lower Vmax, has an Mg2+ concentration of 10^-2 M<ref>{{journal2}}</ref>.  The Km is also larger the upper curve making the higher [Mg2+] more desirable.
 ==Regulation==
 Enolase is found on the surface of a variety of eukaryotic cells as a strong plamingoen-binding receptor and on the surface of hematopietic cells such as monocytes, T cells and B cells, neuronal cells and endothelial cells.  Enolase in muscle cells can bind other glycolytic enzymes, such as phosphoglycerate mutase, muscle creatine kinase, pyruvate kinase, and muscle troponin, with high affinity.  This suggests that enolase helps facilitate muscle contraction by creating a functional glycolytic segment in the muscle where ATP production occurs.  Myc-binding protein (MBP-1) is similar to the α-enolse structure and is found in the nucleus as a DNA-binding protein<ref>{{journal}}</ref>.
-Enolase is regulated by the concentration of Mg2+ and the previous steps of glycolysis.
+Additional levels of enolase regulation occur through the addition of flouride ion, F-. F- forms a complex with Mg2+ already bound at the enzyme's active site. This blocks enolase from binding the substrate 2PG, causing it to build up and thereby slowing the process of glycolysis.
 ==Additional Resources==