BioChem 330 - Course Outline 2011 Part 2

Gly 1 hexokinase: first ATP utilization O P H OH ATP + 2.7.1.1 H O HO HO H H Hexokinase H OH OH Glucose H O O O + ADP H O HO HO H H H OH OH Glucose-6-phosphate * ATP is the second substrate, ADP the second product. * Reaction type is phosphoryl shift: hexokinase catalyzes the transfer of the g phosphate group from ATP to glucose, C6 OH acting as a nucleophile attacks the P of terminal phosphate. What about the sugar cyclization? What about the transition state? What about metals? DGo’ = -16.7 kJ/mole Gly 1 hexokinase, glucokinase: * Hexokinase shows multisubstrate binding, product inhibition, and induced fit. * Km glucose is 0.1 mM, in erythrocytes [glucose] = 5 mM Km/S is <<< 1, enzyme is saturated with glucose and operating at Vmax * Km ATP is 4.0 mM in the absence of glucose --- weak binding Km ATP is 0.1 mM in the presence of glucose --- tighter binding glucose binding increases affinity of enzyme 40 fold for ATP In liver, glucokinase is an isozyme of hexokinase, only binds glucose, only activates at higher glucose levels and doesn’t show product inhibition, precedes storage of glucose as glycogen, a process unique to liver and muscle cells. All Km, S values throughout this handout are taken from Enzyme Structure and Mechanism by Alan Fersht, p256 Gly 1 hexokinase: Induced Fit, Hinge Motion upon Binding Example of Induced Fit: Protein folds over substrates to exclude water, and catalyze reaction by proximity and orientation. Two lobes rotate by 12o resulting in relative movements of 8Å . Ligands that bind but do not induce conformational change are not substrates. Product dissociation is quite slow and rate limiting. Gly 1 yeast hexokinase, structure Substrates bind at domain interface Hinge motion of protein domains: from Gerstein’s Lab at Yale http://bioinfo.mbb.yale.edu/Mol MovDB/ Steitz, Shoham, Bennett Jr.: Structural dynamics of yeast hexokinase during catalysis. Philos Trans R Soc Lond B Biol Sci 293 pp. 43 (1981) Image of hexokinase, PDB coordinates 2e2n.pdb vs 2e2q.pdb are morphed to represent the motion in the protein upon binding. Gly 2 phosphoglucose isomerase O P H O 6 HO 4 HO 3 H O O O O H 2 P 6 CH2 O H O 5 1 Phosphoglucos e isomerase OH OH H 5.3.1.9 * Rxn type is isomerization: gly2 catalyzes the conversion of aldo sugar to keto sugar. OH 2 OH HO H Fructose-6-p hosphate Glucose-6-phosphate * ΔG’ about zero, reaction is almost at equilibrium H2 C 1 O H 5 H OH O R O OH 1 C H 2 R' H R C H OH O Schematically: DGo’ = +1.7 kJ/mole 1 C 2 OH H C R' OH Mechanism of Gly 2: Phosphoglucose Isomerase Mechanism of Gly 2: Phosphoglucose Isomerase What would the free energy profile for this reaction look like? Assume: Km = 100 uM kcat = product dissociation = 103s-1 Activation Barrier = DGtt = [17.6 - 1.36 log kcat] Binding Energy= DGo = = - RTln(1/Km) = - 5 kcal Mechanism of Gly 2: Phosphoglucose Isomerase What would the free energy profile for this reaction look like? Assume: Km = 100 uM kcat = product dissociation = 103s-1 Free Energy Free energy profile: E + S⇌ ESc ⇌ ESo ⇌ EI ⇌ EPo ⇌ EPc →E + P E+S EI E+P EPo EPc ESc ESo DGtt = [17.6 - 1.36 log kcat] DGtt= +12.5 kcal/mole Reaction Coordinate DG = Binding Energy= - RTln(1/Km) = - 5 kcal DGtt =+ 50.0 kJ/mole Gly 2 Mechanism: Enolate formation • Enol formation Acid Base H O H 1 R C C 3 1 R R H ‡ H O O C C R2 R1 3 R C C R3 R2 R2 • Enolate formation B H Ž+ H R1 O O C C R 2 R3 R1 Ž— R R2 O R C C ‡ 3 1 C R2 O R C R 3 1 C R2 C R3 Gly 2 Structure 1 HOX from Rabbit 2.0 Å resolution structure of a dimer from rabbit phosphoglucoisomerase with 2 f6p, one bound in center of each monomer. Gly 3 Phosphofructokinase Second ATP Utilization O O O O O P O CH2 H ATP + H2 C O OH HO Requires Mg+2 H Fructose-6-phosphate O O P O CH2 Phosphofructokinase OH H OH O H2 C O H OH H 2.7.1.11 OH O P O + ADP H HO Fructose-1,6-diphosphate bis Committed (irreversible) step of glycolysis DGo’ = -14.2 kJ/mole highly regulated: + regulators: Pi, AMP, - regulators: ATP, fatty acids, citrate, H+ also deinhibitors of ATP inhibition: F2-6BP (later) Gly 3: Structure of 1PFK from e. coli Gly 4 Aldolase O O H2 C HO H H 1 2 3 4 5 6 H2 C O P O O O 4.1.2.13 H O 2 O 3 H HO Aldolase OH OH O H12 C O P H O O H Fructose-1,6-diphosphate H 4 5 6 H2 C P O O Dihydroxyacetone phosphate P2 O OH O P1 O P O O Glyceraldehyde3-phosphate DGo’ = +23.8 kJ/mole The backward reaction (condensation) is spontaneous. Gly 4 Aldolase Mechanism Important Themes in Gly 4 1. Schiff Base Linkage: • Active site Lysine • Covalently binds the substrate through a Schiff base linkage. • activates substrate for bond cleavage by acting as an electron sink for negative charge that would otherwise build up on C3 Voet, Voet, and Pratt Fundamentals of Biochemistry Gly 4 Aldolase Mechanism 2. Base/Acid Chemistry • tyrosinate at active site initiates C-C bond cleavage by removing a proton from alcohol on C4 • Aldehyde forms on C4, breaking C3-C4 bond • P1-GAP, leaves • tyrosine can donate proton back to C3 • Hydrolysis of bound enamine generated DHAPP2. (O18 water, incorporated into DHAP) Voet, Voet, and Pratt Fundamentals of Biochemistry Gly 4: Aldols, and Enols and Enamines Oh, My! • Enamine formation SHIFF BASE FORMATION R NH2 Lysine O H 1 R R R C C H N 3 R -H2 O H C 1 R C 3 C C R2 Imine R2 R N R1 R ‡ H R1 3 R C H N C R2 Enamine R2 • General Aldol Condensation (Reverse Reaction) H3 C O O O C C C H H2 C C4 C3 P1 P2 H H3 C O H C H2 C OH H R3 H3 C C H O C H2 C H Gly 4 Aldolase Substrate binding Muscle cells Km [FBP] S 100 uM 32 uM Km/S 3.1 cleavage reverse reaction (C-C bond formation) [G3P] 1000 uM 3 uM [DHAP] 2000 uM 50 uM 333 condensation 40 * FBP binds best, Km/S shows about 1/2 Vmax * DHAP, G3P 10X weaker binding, Km/S shows not at all maximized for this direction. * Muscle cell has very little of G3P, this pulls the reaction forward.