BioChem 330 - Course Outline 2011 Part 3

Gly 4 Aldolase Title Fructose 1,6-Bisphosphate Aldolase From Rabbit Muscle Classification Lyase Compound Mol_Id: 1; Molecule: Aldolase; Chain: A, B, C, D; Ec: 4.1.2.13; Biological_Unit: Tetramer Exp. Method X-ray Diffraction 1ADO Active site with Lys41 shown in blue, substrate analogue spacefilled and cpk colored Gly 4 Aldolase 1ADO Active site Tyr 301 shown spacefilled in green Gly 5 Triose Phosphate Isomerase TIM H O T ri ose Isom era se H OH H2 C O H 5.3.1.1 HO H O O P Glyceraldehyde3-phosphate O H2 C O O O P O O Dihydroxyacetone phosphate DGo’ ketone to aldehyde = +7.5 kJ/mole prod/react =1/20 Gly 5 TIM Mechanism O O O O P O CH2 O H O O O CH2 H P HB Triose Isomerase OH O C Glu-165 O H O Triose Isomerase B CH2 H OH HB H O O O O P O H H O O C H O O Glu-165 O C Glu-165 • Enol formation requires both protonation and deprotonation • Two groups involved in proton shuttles: Glu165 and His-95. Gly 5 TIM Kinetics for DHAP • kcat = 2.0 x 105 s-1 (ΔGtt = 10.4 kcal/mole) – modest catalysis • Km = 8.7 x 10-4 M – enzyme binds just well enough (0.87 mM) • kcat/Km = 2.4 x 108 M-1s-1 – virtually the highest possible for bimolecular process of E + DHAP = E + GAP, TIM is the perfect enzyme • Close to saturation? – [S] = [DHAP] = 50 uM – Km = 870 uM – Km/S = 17/1 enzyme not working at Vmax, can go faster if necessary Gly 5 TIM structure • Enzyme Motion – When the enzyme binds its substrate the loop closes over the active site, shielding the substrate from water. The loop appears to close as a rigid lid, stabilized by internal hydrogen bonds. Its motion involves movements of alpha carbons of up to 8 A. The closure involves the filling of a cavity near the base of the helix to which the loop is connected and the formation of new hydrogen bonds and contacts. – Structures 2YPI , 3TIM , 6TIM Gly 5 TIM Structure 2YPI Crystallographic Analysis Of The Complex Between Triosephosphate Isomerase and 2-Phosphoglycolate At 2.5 Å Classification Isomerase (Intramolecular Oxidoreductse) Compound Triose Phosphate Isomerase (TIM) (E.C. 5.3.1.1) Complex With 2Phosphoglycolic Acid Exp. Method X-ray Diffraction Gly 5 TIM Structure 2YPI Glu 165 that acts as a base to abstract a proton is shown in blue at right His95 that participates in acid/base mechanism shown in yellow at left. Phase II 2GAP + 2NAD+ + 4 ADP+ 4 HPO4-2 ---> 2 pyruvate + 2 NADH + 4 ATP + 6 H2O Cashing In, here we see a production of 2 NADH and when we subtract the 2 ATP invested from 4 ATP above, net 2 ATP Note that reaction above isn’t charge or mass balanced, and protons aren’t accounted for directly, some reactions will generate. 6 waters produced, 2 come from substrate in gly 9 and 4 come from gly 7 and gly 10 (ADP-4 + HPO4-2= ATP-6 + H2O+H+) Gly 6: Glyceraldehyde-3-Phosphate Dehydrogenase: 1.2.1.12 O [HPO4]-2 + H H Gl ycera ld ehyde -3 -p hosp hate O de hydrogen ase OH H2 C O O Glyceraldehyde3-phosphate O P O O P O H O OH H2 C O NAD+ NADH O 1,3-diphosphoglycerate + H+ O P O O 2 Free Energy • Note: overall oxidation • Exergonic oxidation of aldehyde drives reaction • Cofactor: NAD+ 0 -2 -4 -6 -8 -10 -12 Start Hexokinase PGI PFK A ldolase TIM G-3-P DH PGKPGM EnolasePK DGo’ = +6.3 kJ/mole Gly 6: Mechanism *C149 attacks GAP, H176 grabs proton *thiohemiacetal intermediate * hydride shift to NAD+ GAP oxidized * second acylthioester intermediate * NADH release is rate limiting *second NAD+ binds * Pi is 2nd substrate *1,3 BPG released This step is where the substrate is oxidized!! Base is H176 Deprotonation of phosphate yields H+ in product Voet, Voet, and Pratt Fundamentals of Biochemistry Gly 6: Saturation? Km and [S] GAP + NAD+ + Pi = 1,3 DPG + NADH Km [S] Km/[S] GAP 70 uM 3 uM 23 less than ½ Vmax NAD+ 46 uM 600 uM 0.08 sat’d w NAD+ Pi high 2,000 uM >10 weak binding •Km/S<< 1 predicts that gly6 is saturated w NAD+ and not sensitive to changes in it, or controlled by its concentrations •GAP and Pi both have Km/S >1, both are >10 for very different reasons. GAP has good binding but a low concentration of substrate. Pi has horrible binding, but a high concentration of substrate. Since the binding won’t change, we say that reaction is controlled by GAP because small changes in its concentration will dramatically alter the rate. Gly 6:Structure 1GAD. * Dimer structure from e.coli * NAD+ binding in Rossman fold * Pos. Cooperativity Hill 2.3 Gly 7: Phosphoglycerate Kinase First ATP Generation 2.7.2.3 O O O O P Phosphoglycerate Ki nase O H OH H2 C O O P OH O O P O O 3-Phosphoglycerate 2 0 Free Energy • Very exergonic , ATP generated • Only monomeric enzyme on pathway • Closely associated with Gly6 -18.5 kJ/mole H ADP ATP 1,3-diphosphoglycerate DGo’ = O H2 C O O O -2 -4 -6 -8 -10 -12 Start Hexokinase PGI PFK A ldolase TIM G-3-P DH PGKPGM EnolasePK Gly 7: Structure 16PK from Trypanosoma brucei * ADP binding site looks like NADH binding site, here crystallized with NADH analogue * ADP binds tightly, more like cofactor, Km/S = 0.05 * 1,3 DPG Km/S = 9 Gly 8: Phosphoglycerate Mutase O O H OH H2 C O O P Phosphogly cerate Mutase O 2.7.5.3 H O O O H2 C O OH P O O O 3-Phosphoglycerate 2-Phosphoglycerate DGo’ = +4.4 kJ/mole 2 Free Energy 0 -2 -4 -6 -8 -10 -12 Start Hexokinase PGI PFK A ldolase TIM G-3-P DH PGKPGM EnolasePK Gly 8: PGM Mechanism *Base (Lys?) interacts with carboxylic acid of 3PG for binding * Active enzyme phosphorylated by 2,3-DPG *His His duo at active site *Km varies from 240 uM in brain to 5000 uM in muscle. (nature of base?) *[S] 3PG 40-60 uM *Km/S = 6-80, E not sat’d with S, room to speed up. Lys Voet, Voet, and Pratt Fundamentals of Biochemistry Gly 8: 3PGM structure from Schizosaccharomyces pombe • Gly 8 has a relatively simple structure with a beta twist and helical unit, 3PG binds at interface. • Two his 4 Å apart shown in blue wireframe • His adjacent to C2 has Pi, His adjacent to C3 extracts Pi from C3 while OH on C2 extracts Pi from Pi-His