Energy storage in cells Josef Fontana EC - 58
Overview of the lecture Introduction to the storage substances of human body Overview of storage compounds in the body Glycogen metabolism Structure of glycogen Synthesis and degradation of glycogen Phosphorylation and dephosphorylation as a regulatory mechanism of the glycogen metabolism Synthesis of fatty acids and TAG Differences between synthesis and degradation of fatty acids How works the fatty acid synthase Elongation and desaturation of fatty acids Synthesis of TAG
Introduction to the storage substances of human body Overview of storage compounds in the body
Overview of storage compounds TAG Glycogen No storage protein in the body TAG are excelent for energy storage - 1g of fat has 6 times more energy than 1g of hydrated glycogen Complete oxidation of 1g of FA = 38 kj Complete oxidation of 1g of saccharides or proteins only 17 kj
Overview of storage compounds 70 kg man has: in the body 1) 420 000 kj in TAG 2) 10 000 kj in proteins (muscle) 3) 2 500 kj in glykogen 4) 170 kj in glucose Glycogen and glucose are sufficient to supply the body one day, TAG many weeks
Glycogen metabolism Structure of glycogen
Glycogen Animal saccharide storage In liver (100g), skeletal muscle (500g) and in small quantities in each cell 1) liver glycogen: to maintain glycemia 2) muscle glykogen: for internal muscle use
Glycogen structure Branched homopolymer Most residues bound by α 1 4 bonds Branching: α 1 6 bond These branches are extended by α 1 4 bond
Glycogen has two ends Only on the non-reducing ends can take place reactions (lengthening or shortening) Reducing end is the one with the hemiacetal hydroxy group - bound to tyrosine in glycogenin
Glycogen metabolism Synthesis and degradation of glycogen
Glycogenesis (glycogen synthesis) Cytosol Glucose phosphorylation to Glc-6-P: glucokinase in liver and hexokinase in muscle Isomeration of Glc-6-P Glc-1-P: phosphoglucomutase Glc-1-P reacts with UTP UDP-Glc (activated Glc, bond on C1): Glc-1-P uridylyltransferase UDP-Glc is bound to the non-reducing end of glycogen: glycogen synthase
Glycogen synthase Binds UDP-Glc to the non-reducing end of glycogen UDP is released Chain of glucose molecules lengthens, until it reaches a certain length and branching occurs
Branching enzyme Removes oligosaccharide (6-7 Glc residues) from growing chain and adds it to a hydroxy group on the C6 in Glc Forms α 1 6 bond These branches are extended by glycogen synthase Branching enzyme = amylo-(1,4 1,6)- transglycosylase
Regulation of glycogen synthesis Glycogen synthase is regulated by phoshorylation: phosphorylation inactivates dephosphorylation activates Insulin activates Glucagon and adrenaline inhibit
Glycogenolysis Cytosol 1) Phosphorolytic cleavage (inorganic phosphate is used): glycogen phosphorylase Glc-1-P (Cori ester) 2) Isomeration of Glc-1-P to Glc-6-P: phosphoglukomutase
Cutting branches off Degradation of glycogen stops at the 4th Glc before the branching point Glucanotransferase (glycosyltransferase) transfers three glucose residues from the 4- residue glycogen branch to the main chain Only one glucose molecule remains (α 1 6 bond) cleaved by debranching enzyme (amylo-α1 6-glucosidase) Linear glycogen chain glycogen phosphorylase
Regulation of glycogenolysis Glycogen phosphorylase is activated phosphorylated Phosphorylase kinase Insulin inhibits Counter-regulatory hormones activate
Synthesis of fatty acids and TAG Differences between synthesis and degradation of fatty acids
Differences between synthesis and degradation of fatty acids FA synthesis in cytosol, degradation in matrix Intermediates of FA synthesis are bound to ACP (acyl carrier protein), intermediates of degradation bound to CoA Enzymes of FA synthesis form one big multienzyme complex - Synthase of FA, degradation enzymes are free in matrix
Differences between synthesis and degradation of fatty acids FA chain is extended by 2 carbon atoms from AcCoA activated substrate is malonyl~coa Reducing cofactor for synthesis is NADPH, oxidising cofactors for degradation are FAD and NAD +
Differences between synthesis and degradation of fatty acids FA synthesis (on FA synthase) ends with palmitate (C 16 ) Further chain elongation and formation of unsaturated acids catalyse other enzymes
Synthesis of fatty acids and TAG Synthesis of malonyl~coa
Synthesis of malonyl~coa Substrate for FA synthesis: AcCoA Carboxylation to malonyl-coa AcCoA + ATP + HCO 3 - malonyl~coa + ADP + P i + H + AcCoA carboxylase (biotin vitamin H or B7) Regulatory enzyme CO 2 removed during condensation with growing FA
Synthesis of fatty acids and TAG How works the fatty acid synthase
Mammalian fatty acid synthase Homodimer of 2 identical subunits (260 kda) Each subunit has three domains connected by moving regions: 1) domain 1 substrates entry and condensing unit - both transferases and condensing enzyme - CE 2) domain 2 reduction unit includes ACP, β- ketoacylreduktase, dehydratase and enoylreductase 3) domain 3 thioesterase cleaves palmitate
Mammalian fatty acid synthase Places where are bound intermediates on FA synthase: 1) thiol group of cysteine (CE) 2) thiol group of phosphopantetheine (bound to serine in ACP)
Steps of FA synthesis 1. Synthesis of malonyl-coa: acetyl-coa carboxylase 2. Reaction AcCoA + CE: acetyltransacylase 3. Reaction malonyl-coa + ACP: malonyltransacylase 4. Condensation reaction: condensing enzyme Acetyl-CE + malonyl-acp acetoacetyl-acp + CE + CO 2
Steps of FA synthesis 5. First reduction: β-ketoacylreductase Acetoacetyl-ACP + NADPH + H + D-3- hydroxybutyryl-acp + NADP + 6. Dehydration: 3-hydroxyacyldehydratase D-3-Hydroxybutyryl-ACP crotonyl-acp + H 2 O 7. Second reduction: enoylreductase Crotonyl-ACP + NADPH + H + butyryl-acp + NADP +
FA synthase works as a dimer Condensation between malonyl-acp (one subunit) and acetyl-ce (second subunit) S H 3 C CE C S O C H 2 C O C O - ACP SH O SH KONDENZACE CO 2 HS CE H 2 C C S ACP SH CH 3 C O SH O New acyl remains on ACP ACP CE ACP CE
First reduction H 3 C O C C H 2 O C S ACP REDUKCE H 3 C HO C H C H 2 O C S ACP Acetoacetyl-ACP H + + NADPH NADP + D-3-Hydroxybutyryl-ACP
Dehydration HO H O DEHYDRATACE H O H 3 C C C H 2 C S ACP D-3-Hydroxybutyryl-ACP H 2 O H 3 C C C C S H Krotonyl-ACP ACP
Second reduction H 3 C O H C C C S H Krotonyl-ACP ACP REDUKCE H + + NADPH NADP + H 3 C H 2 C C H 2 O C Butyryl-ACP S ACP
Process continues Change of subunits after one rotation Palmitate (C16) is an end product Thioesterase cleaves palmitate from ACP - hydrolysis of the thioester bond with phosphopantetheine
Palmitate synthesis requires 8 AcCoA, 14 NADPH a 7 ATP AcCoA produced in matrix inner mitochondrial membrane is impermeable transport via citrate 8 NADPH from the citrate transport to cytosol and remaining 6 NADPH in pentose cycle
Citrate as AcCoA bearer High level of citrate in matrix transport to cytosol cleavage by ATP-citrate lyase: Citrate + ATP + HSCoA + H 2 O AcCoA + ADP + P i + OAA AcCoA and OAA have different fate in cytosol
OAA returns to matrix Inner mitochondrial membrane is impermeable to OAA Reduction of OAA to malate by cytosolic malate dehydrogenase: OAA + NADH + H + malate + NAD + Oxidative decarboxylation of malate by NADP + -malate enzyme (malic enzyme): Malate + NADP + Pyr + CO 2 + NADPH
OAA returns to matrix Pyruvate transport to matrix carboxylation by pyruvate carboxylase: Pyr + CO 2 + ATP + H 2 O OAA + ADP + P i + 2 H + Summary equation: NADP + + NADH + ATP + H 2 O NADPH + NAD + + ADP + P i + H +
Regulation of FA synthesis Enough substrates (saccharides/aa) and energy AcCoA-carboxylase: 1) insulin activates 2) glucagon and epinephrine inhibit 3) citrate activates 4) inhibition by palmitoyl-coa feedback inhibition 5) AMP inhibits
Synthesis of fatty acids and TAG Elongation and desaturation of fatty acids
Synthesis of other fatty acids Chain elongation elongases Synthesis of unsaturated FA desaturation desaturases ER membrane
Desaturation Mammals lack enzymes catalyzing formation of the double bond further than on C9 New double bonds are always formed between the existing double bond and a carboxyl group Mammals can not synthesize linoleic (18 : 2 cis D 9, D 12 ) and linolenic (18 : 3 cis D 9, D 12, D 15 ) acid both eare essential
Synthesis of fatty acids and TAG Synthesis of TAG
Synthesis of TAG