Lecture 20 Entering the mitochondrion PDF

Changes in atmospheric oxygen over time Pasteur point (0.3%) 15 Only a Small Amount of Energy Available in Glucose Is Captured in Glycolysis Glycolysis...

Changes in atmospheric oxygen over time Pasteur point (0.3%) 15 Only a Small Amount of Energy Available in Glucose Is Captured in Glycolysis Glycolysis 2 G′ = –146 kJ/mol Glucose Full oxidation (+ 6 O2) 6 CO2 + 6 H2O G′ = –2,840 kJ/mol 16  Cellular Respiration Evolutionary origin: developed about 2.5 billion years ago Microorganisms, plants, animals Three major stages: - acetyl CoA production - acetyl CoA oxidation - electron transfer and oxidative phosphorylation 17 Catabolic Fates of Pyruvate 18 Pyruvate oxidised in Mitochondria Mitos = thread, Chondros = granule (Carl Benda, 1898) 19  Respiration: Stage 1 Acetyl-CoA Production Generates ATP, NADH, FADH2 Carbohydrates release 1/3 of total potential CO2 during Stage 1. 21 Mitochondrial Pyruvate Carrier (MPC) Transport inhibited by acetylation of MPC 22  Entry of Pyruvate Into Mitochondria diffuses from cytosol through porins - outer membrane (OMM) transported across inner mitochondrial membrane (IMM) into matrix - mitochondrial pyruvate carrier (MPC) Encoded by two genes (MPC1 and MPC2) MPC1 and MPC2 mutated in a high proportion (80%) of some cancers, - gliomas (brain glial cell tumours) Mutations reduce proportion of cytosolic pyruvate that undergoes oxidation via Krebs’ Cycle - Warburg effect? 23 - Pharmacological inhibitor 24  Conversion of Pyruvate to Acetyl-CoA Pyruvate dehydrogenase complex in mitochondrial matrix – oxidative decarboxylation of pyruvate – requires 5 cofactors, 3 types of enzymes and at least 60 subunits in highly ordered structure – TPP, lipoate, and FAD are prosthetic groups. – NAD+ and CoA-SH are co-substrates. Thiamine (B1) Riboflavin (B2) Niacin (B3) Pantothenate (B5) (CoA) 25  Pyruvate Dehydrogenase Complex (PDC) PDC is a large (up to 10 MDa) multienzyme complex. - pyruvate dehydrogenase (E1) - dihydrolipoyl transacetylase (E2) - dihydrolipoyl dehydrogenase (E3) Advantages : - Short distance between catalytic sites: rate enhancement. - Substrate Channeling minimizes side reactions. - Regulation of one subunit affects entire complex. 26 E coli PDH complex units Prosthetic Groups Pyruvate decarboxylase 24 Thiamine pyrophosphate Dihydrolipoyl transacetylase 24 lipoamide (and CoA-SH) Diydrolipoyl dehydrogenase 12 flavin adeninine dinucelotide (FAD) and NADH Plus 2 regulatory proteins/enzymes – protein kinase and phosphoprotein phosphatase. Lipoyl domain of E2 27  Structure of Lipoyllysine Prosthetic group strongly bound to E2 protein. Lipoic acid covalently linked to E2 via lysine residue 28 Overall Reaction of PDC 29  Sequence: Oxidative Decarboxylation of Pyruvate Enzyme 1 (E1) Step 1: Decarboxylation of pyruvate, hydroxyethyl derivative on TPP, -release of CO2 Step 2: Oxidation of hydroxyethyl to acetyl group, lipollysyl group of E2 ‒ Electrons reduce lipoamide and form a thioester. Enzyme 2 (E2) Step 3: transfer acetyl group to CoA to form acetyl-CoA (product 1) Enzyme 3 (E3) Step 4: Reoxidation of the lipoamide cofactor Step 5: Regeneration of oxidized FAD cofactor, forms NADH (product 2) 30 E3 subunit of PDC is same as αKGDH 31  Regulation of PDH Allosteric and covalent mechanisms Strongly inhibited by product (ATP, acetyl-CoA and NADH) Allosteric inhibition enhanced in presence of long-chain fatty acids Activated by AMP, CoA and NAD+ 32  Regulation of PDH Reversible phosphorylation of serine (S163) on E1 – phosphorylation: inactive – dephosphorylation: active PDH kinase and PDH phosphatase - part of mammalian PDH complex. – Kinase is activated by ATP, NADH and Acetyl Co-A. high ATP → phosphorylated PDH → less acetyl-CoA low ATP → kinase less active, phosphatase removes Pi from E1 → more acetyl-CoA 33  Mutations in genes for PDH – lactate acidosis - weakness Thiamine deficiency results in Beriberi, a disease characterized by loss of neural function (brain can’t oxidize pyruvate) - occurs primarily in populations that rely on white (polished) rice - lacks the hulls where most thiamine found 34 35 Previous lecture: Alzheimer’s. A-resistant nerve cells exhibit increased glucose uptake and glycolytic activity Glucose Consumption Lactate production 36 Soucek et al. Neuron 39:43 (2003); Newington et al. PLoS One (2011) Overexpression of PDK1 in B12 cells confers resistance to multiple apoptotic stimuli 20 M A 200 M H2O2 300 ng/ml Staurosporine 37 Newington et al. J. Biol. Chem (2012)