Conversion of Glucose - Biochemistry - Lecture Slides, Slides of Biochemistry

Download Conversion of Glucose - Biochemistry - Lecture Slides and more Slides Biochemistry in PDF only on Docsity! Glycolysis Docsity.com Glycolysis The conversion of glucose to pyruvate to yield 2ATP molecules •10 enzymatic steps •Chemical interconversion steps •Mechanisms of enzyme conversion and intermediates •Energetics of conversions •Mechanisms controlling the Flux of metabolites through the pathway Docsity.com Inhibitors were used. Reagents are found that inhibit the production of pathway products, thereby causing the buildup of metabolites that can be identified as pathway intermediates. Fluoride- leads to the buildup of 3-phosphoglycerate and 2-phosphoglycerate 1940 Gustav Embden, Otto Meyerhof, and Jacob Parnas put the pathway together. Docsity.com Pathway overview 1. Add phosphoryl groups to activate glucose. 2. Convert the phosphorylated intermediates into high energy phosphate compounds. 3. Couple the transfer of the phosphate to ADP to form ATP. Stage I A preparatory stage in which glucose is phosphorylated and cleaved to yield two molecules of glyceraldehyde-3- phosphate - uses two ATPs Stage II glyceraldehyde-3-phosphate is converted to pyruvate with the concomitant generation of four ATPs-net profit is 2ATPs per glucose. Glucose + 2NAD+ + 2ADP +2Pi  2NADH + 2pyruvate + 2ATP + 2H2O + 4H + Docsity.com Oxidizing power of NAD+ must be recycled 1. Under anaerobic conditions in muscle NADH reduces pyruvate to lactate (homolactic fermentation). 2. Under anaerobic conditions in yeast, pyruvate is decarboxylated to yield CO2 and acetaldehyde and the latter is reduced by NADH to ethanol and NAD+ is regenerated (alcoholic fermentation). 3. Under aerobic conditions, the mitochondrial oxidation of each NADH to NAD+ yields three ATPs NADH produced must be converted back to NAD+ Docsity.com Hexokinase reaction mechanism is RANDOM Bi-Bi Glucose ATP ADP Glu-6-PO4 When ATP binds to hexokinase without glucose it does not hydrolyze ATP. WHY? The binding of glucose elicits a structural change that puts the enzyme in the correct position for hydrolysis of ATP. Docsity.com The enzyme movement places the ATP in close proximity to C6H2OH group of glucose and excludes water from the active site. There is a 40,000 fold increase in ATP hydrolysis upon binding xylose which cannot be phosphorylated! O OH H OH OHH H H a-D-Xylose Docsity.com Yeast hexokinase, two lobes are gray and green. Binding of glucose (purple) causes a large conformational change. A substrate induced conformational change that prevents the unwanted hydrolysis of ATP. Docsity.com 2 Ht ——— ring opening Glucose-6-phosphate (G6P) exchange of 1 G6P H with F6P medium 5 oa cis-Enediolate a it e ~0;POCH, -O C—OH 4 a intermediate H* H HO ring H closure Docsity.com Fructose-6-nhosnhate (F@P) Phosphofructokinase Fructose-6-PO4 Fructose-1,6-bisphosphate 1.) Rate limiting step in glycolysis 2.) Irreversible step, can not go the other way 3.) The control point for glycolysis OH CH2OH H OH H H O -2 O3POCH2 HO OH CH2OPO3 -2 H OH H H O -2 O3POCH2 HO + ATP + ADP Mg++ Docsity.com Aldolase CH2OPO3 -2 C O C C C HHO OHH OHH CH2OPO3 -2 CH2OPO3 -2 C O C HHO H C OHH CH2OPO3 -2 H O + Dihydroxyacetone phosphate (DHAP) Glyceraldehyde-3- phosphate (GAP) Fructose -1,6-bisphosphate (FBP) Aldol cleavage (retro aldol condensation) Docsity.com 1 CH,OH Dihydroxyacetone phosphate (product 2) Fructose-1,6-bisphosphate 1 1,0 Schiff base substrate hydrolysis rem mesyens binding CH,OPO} NH-(CH,, CH,OPO! I HO— C =H, pros ° Enzyme-product Enzyme substrate protonated Schiff base complex tautomerization protonated Schiff # 2 protonation base formation 1,0 CH,OPO} u H—(CH,), CH. OPO} °) co HH — (CHy), HO’ Hy C Idol clea HO! H i aldol cleavage a — H-C7O>H' 38 Le H—C—OH Be i? CH,OPO}- I H—C—OH CH,OPO}- Enamine Glyceraldehyde- Enzyme-substrate interm 3-phosphate protonated Schiff base (product 1) Docsity.com Aldolase is very stereospecific When condensing DHAP with GAP four possible products can form depending on the whether the pro- S or pro R hydrogen is removed on the C3 of DHAP and whether the re or si face of GAP is attacked. CH2OPO3 2- HHO OHH OHH CH2OPO3 2- O CH2OPO3 2- OHH OHH OHH CH2OPO3 2- O D-Fructose 1,6 bisphosphate D-Psicose 1,6 bisphosphate CH2OPO3 2- HHO HHO OHH CH2OPO3 2- O CH2OPO3 2- OHH HHO OHH CH2OPO3 2- O D-Tagatose 1,6 bisphosphate D-Sorbose 1,6 bisphosphate Docsity.com Triosephosphate isomerase DHAP GAP     96 1 10x7.4 DHAP GAP K 2eq   TIM is a perfect enzyme which its rate is diffusion controlled. A rapid equilibrium allows GAP to be used and DHAP to replace the used GAP. Docsity.com 95 O 4H Oo / \ & N\.F HN N Glu_("_ Cc Glu _ 165 Cx, aT a 3 165 0---H—C— 07 | 3CH,0P03- GAP-TIM Michaelis complex DHAP-TIM Michaelis complex His e 8 1 =e Glu_“- a, a 1650 _H a pot G—- 0-H c i 0---H---C—O \=N Glu —c” - be ge-H NZ | 5 Ce C=O ae 2- oO CH,OPO? CH,OPO?- Transition state Transition state oO H oO : p Se His Glu —c” Cc a QU 195 165 \ 4 I 4 ON o— —o—-H”\_| CH,OPO3- & Enediol (or enendiolate) intermediate Docsity.com Geometry of the eneolate intermediate prevents formation of methyl glyoxal Orbital symmetry prevents double bond formation needed for methyl glyoxal Docsity.com Glyceraldehyde-3-phosphate dehydrogenase The first high-energy intermediate Uses inorganic phosphate to create 1,3 bisphosphoglycerate C CH2OPO3 2- OHH HO C CH2OPO3 2- OHH OPO3 2- O + NAD+ + Pi + NADH Docsity.com Mechanistic steps for GAPDH 1. GAP binds to enzyme. 2. The nucleophile SH attacks aldehyde to make a thiohemiacetal. 3. Thiohemiacetal undergoes oxidation to an acyl thioester by a direct transfer of electrons to NAD+ to form NADH. 4. NADH comes off and NAD+ comes on. 5. Thioester undergoes nucleophilic attack by Pi to form 1,3 BPG. The acid anhydride of phosphate in a high energy phosphate intermediate Docsity.com Arsenate uncouples phosphate formation C CH2OPO3 2- OHH OO As O O O C CH2OPO3 2- OHH O - O As OO O O C CH2OPO3 2- OHH HO + GAP DH As OO O O + 3PG Docsity.com