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Chorismate Mutase – an Introduction to Biological Pericyclic Reactions

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Chorismate Mutase – an Introduction to Biological Pericyclic Reactions Vijay Yanamadala Chemistry 27: The Organic Chemistry of Life Triose Phosphate Isomerase – At the Core of Glycolysis Glycolysis Glycolysis is one of the most conserved pathways in all of evolution because of its centrality to the generation of cellular energy. Glucose is of course the starting material for the pathway, and OH OH O through a series of chemical conversions, it generates ATP for use in 3- O4PO cellular processes. Glucose is an aldose, meaning that is has a terminal aldehyde. After glucose intake, it is phosphorylated by expending a OH OH Glucose 6-phosphate molecule of ATP becoming glucose-6-phosphate. This is then converted to the ketose form, called fructose. The conversion involves an enediol intermediate. Draw out the mechanism for practice (it is an important OH OH mechanism that you should know!) Fructose is then further 3- O4PO 3- phosphorylated by expending another molecule of ATP to become OPO4 fructose 1,6-bisphosphate shown to the left. All sugars can take both a OH O cyclic form and an acyclic form. The acyclic form of fructose 1,6- Fructose 1,6-bisphosphate bisphosphate is shown to the left, and the cyclic form is shown below, along with the mechanism for interconversion. Know this simple acid-catalyzed mechanism. Both forms are observed in the cell, although it is the linearized form which undergoes further steps of the glycolysis pathway. OPO 3- OPO 3- OPO 3- H 4 H 4 H 4 OH OH O H O OH H 3-O PO HO H HO H HO H 4 3- HO HO HO OPO4 OH 3- OH 3- OH 3- OPO4 OPO4 OPO4 OH OH H H H After linearization of the fructose 1,6-bisphosphate, it undergoes a reverse aldol reaction to give two isomeric interconvertible triose sugar molecules, called Glyceraldehyde 3’-Phosphate (GAP) and Dihydroxyacetone Phosphate (DHAP). It is the role Triose Phosphate Isomerase (TIM) to interconvert these two, and this process is discussed in detail below. After this, GAP is further metabolized. Fructose Diphosphate Aldolase OH OH OH Fructose Diphosphate Aldolase (FDA) 3- O4PO 3-O PO is the enzyme which performs the 4 OPO 3- 3- 4 HO OPO4 reverse aldol reaction yielding two OH OH O OH Glyceraldehyde 3'-Phosphate(GAP) triose molecules from fructose 1,6- bisphosphate as shown here. The arrow-pushing mechanism is very simple and shown above. The enediol above is protonated, OH yielding DHAP. One interesting OH OH 3-O PO 3- 4 3- OPO4 OPO4 question is whether the enediol O O H H H above is indeed cis (E) as shown O H O 3- 3- above. We can do a Newman H OPO4 HO OPO4 O H O H OPO 3- O H 4 Projection to determine what will OH be the most stable conformation. Shown to the right is the most stable conformation of the H transition state. Remember that the breaking bond must be oriented to have good overlap with the carbonyl antibond HO OPO 3- HO OPO 3- 4 4 (C=O π*), and this is achieved with a ~109° angle (the OH O Dunitz-Burgi Trajectory). Hydrogen bonding positions the Dihydroxyacetone Phosphate (DHAP) 1 Vijay Yanamadala Chemistry 27: The Organic Chemistry of Life carbonyl in a syn orientation with the two alcohols as shown and indeed this yields the E-enediol that is observed. Why is this important, you may ask. Consider that the transition state in TIM for the conversion of DHAP to GAP is an E-enediol and think about enzyme-to-enzyme transfer of intermediate (called enzymatic intermediate transfer). Here is a beautiful example of how direct chemical analysis such as that shown above can lead to a testable biological hypothesis. Indeed, if you search the literature, you will find information on intermediate transfer between FDA and TIM! Triose Phosphate Isomerase (TIM) – A Most Efficient Enzyme OH HR HS The equilibrium shown to the 3- C left is favorable to DHAP, O4PO 3- O4PO OH which is present at a concentration 20 times greater O O than that of GAP in the Glyceraldehyde 3'-Phosphate (GAP) Dihydroxyacetone Phosphate (DHAP) cytoplasm of a cell. However, GAP is required for the final production of pyruvate in glycolysis and is the active substrate. Thus, TIM is a necessary equilibrase which quickly reequilibrates the system so as to ensure a constant supply of GAP for the progress of glycolysis. TIM is one of the most efficient enzymes known – it is only limited by the rate of diffusion of its substrates! TIM gives us full appreciation for the chirality of enzymes and the consequent asymmetry of enzyme catalyzed processes. The first appreciation is that the enzyme active site only allows for the formation of an E-enediol intermediate. This results from the orientation of the hydrogen bonding amino acid residues in the active site. DHAP has two enantiotopic hydrogens that behave differently in chiral environments, such as the active site of TIM. Only the Pro-R hydrogen is deprotonated by the Glutamate-165 of the enzyme! Further this proton is then captured by the Re-face of DHAP. Once again, labeling one of the two enantiotopic hydrogens on the methylene interjecting between the carbonyl and free hydroxyl of DHAP (labeled in red above) allows us to carefully analyze and verify the activity of TIM. If we replace the pro-R hydrogen with a Deuterium in DHAP, the deuterium will migrate to the 2-carbon of GAP. If we replace the pro-S hydrogen with a Deuterium in DHAP, the deuterium will remain attached to the 1- carbon of GAP and will be the aldehyde proton. Glutamate-165 swivels so that it can position the proton over one of the two carbons, yielding either DHAP or GAP. This confirms the fact that TIM is simply an equilibrase! Further, enzymatic catalysis is achieved by stabilizing the positive charge through protonation by Histidine-95, which also deprotonates the ketone or aldehyde after re-tautomerization. Once again, the phosphate serves for molecular recognition and forms a salt-bridge with positively charged Lysine-13. Further Suggested Reading: Bugg Chapter 7 (pages 158 – 162), Chapter 4 (pages 60 – 67). Knowles, J. “Enzyme Catalysis: Not Different, Just Better.” Nature 1991, Mar 14. (350) – Available on page 201 of the Source Book. 2 Vijay Yanamadala Chemistry 27: The Organic Chemistry of Life DHQ Synthase – A Sheep in Wolves’ Clothing The Industrious Use of Substrate Analogs DHQ Synthase – The Overview of the Pathway NAD HB DHQ synthase is part of the H HO O O H B HO O O O O Step 1 Step 2a shikimic acid pathway, a H O O O O O O O O O O pathway used in the OH H P O OH H P O OH H P O O O biosynthesis of the aromatic O O O O amino acids Phenylalanine, Step 2b Tyrosine, and Tryptophan. H--NAD H HB DHQ synthase catalyses a 5 HO O HO HO O O Step 4a O Step 3 step process that converts OH OH O O O O O O O 3deoxyarabinoheptulosonat O H P O O O O H e-7-phosphate (DAHP) into B H O Step 4b 3-dehydroquinate (DHQ). HO O HO Step 5 O Shown to the left is the OH OH O O O O progress of the enzyme- O OH catalyzed reaction. The + BH enzyme is NAD dependent and involves the oxidation and re-reduction of the 5’-hydroxyl. Step 1 is the oxidation of the alcohol to the corresponding ketone, which allows for enolization in step 2a. It is important to note that the phosphate acts as the base for deprotonation in this step, and this was proven through the use of substrate analogs. The enol then undergoes E1cb type elimination of the phosphate in step 2b. Since the only purpose of the ketone was the catalysis for the deprotonation through conjugation, it is at this stage rereduced using the now reduced form NADH which acts as the hydride donor. At this point, a basic residue in the enzyme catalyzes ring opening at the hemiacetal, leading to the acyclic enolate seen in step 4a. This enolate then rotates as seen in step 4b, now positioned for direct attack on the ketone. The enolate attacks, closing the ring, giving us DHQ. The step-by-step analysis of this process was done through the use of transition state analogs,which are discussed in detail here. Step 1 – Does the Oxidation Actually Occur? H O This analog of the natural substrate O + O H NAD O replaces the phosphate with a O O O O phosphonate, which is not a leaving OH H P O OH H P O group (the breaking of a carbon-carbon O O O O bond is required!). Thus, once step 1 is complete, Step 2a is feasible, but the substrate undergoes no further reaction. Thus an equilibrium is established between the keto and hydroxy forms of the analog, and both can be observed in experimentation. This demonstrates that oxidation does indeed occur. Step 2 – How do we know the Phosphate Acts as the Base? H H Well, the first step is showing that it is O H D O even feasible. We realize that there are OH OH O O two prochiral hydrogens on the O O O methylene group attached to the D H H P O H O H O 3 Vijay Yanamadala Chemistry 27: The Organic Chemistry of Life phosphate. Substituting one for deuterium allows us to determine the stereochemistry of the final product.
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