control of metabolic reactions making +ve  G o ‘ reactions happen points of control:  G o ‘ and equilibrium multi-active enzymes: enzyme complexes and multiple active sites

reactions with +ve  G o ‘ can occur by: coupling with a reaction with –ve  G o ‘ –ve physiological  G due to cellular low ratio [products]/[reactants]

1. reactions with +ve  G o ‘ occur by coupling with a reaction with -ve  G o ‘ Thus, ATP  ADP +Pi (  G 0) glucose-6-P + ADP hexokinase Glucoseglucose-6-P + H 2 0  G = 13.8 kJ.mol -1 ATP +H 2 0 ADP +Pi  G = kJ.mol -1 Glucose + ATP overall  G = kJ.mol -1

2. Recall:  G o ' of a reaction may be positive, and  G negative, depending on cellular concentrations of reactants and products. any  [products] or  [substrate] that moves the reaction away from equilibrium ratio causes reaction to proceed spontaneously forward to restore equilibrium Many reactions for which  G o ' is positive are spontaneous in vivo because other reactions cause  [products] or  [substrate].

At equilibrium, no net change so  G = 0. free energy change is related to the equilibrium constant ( K' eq ) = the ratio of [products]/[reactants] at equilibrium I won’t be asking you to solve any of these equations!

many reactions are near equilibrium then  G ~0 (no net change in free energy) easily reversed by changing ratio of [products]/[substrate]  as don’t need to overcome high  G For A+B ↔C+D  product A+B  C+D  substrate A+B  C+D enzymes that catalyse such reactions act to restore equilibrium rate regulated by [products]/[reactants]

Implication: a reaction near equilibrium may have +ve  G o ' but be spontaneous in the cell   ve  G because other reactions cause  [products] or  [substrate].

Other reactions are FAR from equilibrium enzyme rate is too slow to allow products to build to equilibrium concentration [substrate] builds up in excess of K eq   G <<<0 (highly negative) not affected by  [substrate] (saturated) essentially irreversible rate controlled by changing activity of enzyme (eg allosteric interactions) reactions with  G <<0 are often sites of regulation

1. Often occur early as a “committed step” in metabolic pathways (eg AcetylCoA carboxylase) 2.most metabolic pathways are irreversible ≥ 1 step with -ve  G required to drive: eg PDH, pyruvate carboxylase ) one way street: return by a different street  3. catabolic and anabolic pathways are separate  independent control (eg glycolysis and gluconeogenesis ( eg pyruvate carboxylase ) use different enzymes) reactions with  G <<0 are often sites of regulation

Pyruvate dehydrogenase a pretty, pink multi-enzyme complex ‘gatekeeper’ to entry to citric acid cycle

pyruvate dehydrogenase 6 NADH 2 NADH Pyruvate dehydrogenase regulates entry into the citric acid cycle of metabolites leaving glycolysis

Summary 1.structure of PDH complex 3 enzymes (E1, E2, E3) 2.reactions of PDH complex 5 reactions, 5 cofactors 3.mechanism of PDH complex lipoamide swinging arm 4.regulation of PDH complex de/phosphorylation of E1 product inhibition of E2 and E3 Excellent animation of PDH reactions if you can access it: (not examinable, but might help understanding!)

3 different ENZYMES 5 COFACTORS (non-covalently associated) E1: pyruvate dehydrogenase +TPP E2: dihydrolipoyl transacetylase + lipoamide E3: dihydrolipoyl dehydrogenase + FAD + NADH +CoenzymeA PDH = multi-enzyme complex 5 sequential reactions catalyse

overall….. pyruvateAcCoA NAD + NADH CoA CO 2 high energy bond irreversible (3C) (2C)

multi-enzyme complex (E. coli) a) dihydrolipoyl transacetylase (E 2 )  arranged as corners of a core cube surrounded by an outer cube: b) pyruvate dehydrogenase (E 1 )  edges c) dihydrolipoyl dehydrogenase (E 3 )  faces Note that there are many copies of each enzyme in each complex

PDH structure is more complex in other organisms dodecahedron core= 12 pentagon faces 20 vertices (E 2 trimers) in mammals = + kinase + phosphatase E 2 core of B. stearothermophilus

each enzyme uses a cofactor 2 lipoate binding domains in each E2 FAD in each E3 TPP in each E1

5 sequential reactions pyruvate dehydrogenase (E 1 ) dihydrolipoyl transacetylase (E 2 ) dihydrolipoyl dehydrogenase (E 3 ) In summary: 1) pyruvate is decarboxylated  hydroxyethyl, requires TPP to stabilise the intermediate. 2) hydroxyethyl oxidised to acetyl, collected by lipoamide of E2, which gets reduced. 3) lipoamide of E2, passes acetyl to coenzyme A  acetyl CoA. 4) lipoamide of E2, gets re-oxidised, gives its electrons to FAD in E3 which 5) passes electrons to NAD  NADH

1. decarboxylation by E 1 Pyruvate hydroxyethyl- loss of CO 2  conversion of pyruvate to a 2 carbon moiety E1 has a bound coenzyme (TPP) that attacks pyruvate and stabilises the intermediate (3C) (2C)

i. TPP forms a carbanion H + readily dissociates (due to adjacent N + ) N + stabilises the carbanion H+

 releases CO 2 CO 2 ii. nucleophilic attack by TPP carbanion on electron-deficient C2 of pyruvate hydroxyethyl-TTP

iii. TTP stabilises the carbanion intermediate after CO 2 is lost. CO 2 can’t just remove CO 2  highly unstable intermediate I won’t ask you to recreate bond rearrangements!

- R - lys 2. formation of acetyl by E 1 REDUCTION gain of hydrogen dihydro- lipoamide OXIDATION hydroxyethyl acetyl- lipoamide - R - lysine + TTP regenerated E2E2 hydroxyethyl is transferred the lipoamide group of E2, Lipoamide (= lipoic acid linked covalently to Lysine) contains a cyclic disulfide reactive group that can be reversibly reduced  dihydro-lipoamide

E 2 uses lipoamide as a cofactor lipoic acid acts as a long flexible arm that can transfer substrates between active sites there are actually 2 lipoate-binding domains in each E 2.  cyclic disulphide reversibly reduced and oxidised  lipoamide = lipoic acid covalently bound to lysine in E2

3. trans-esterification acetyl group transferred by E 2 to CoA = high energy thioester bond

Form between carboxylic acid (COOH) and a thiol (SH) eg thiol in CoenzymeA eg Acetyl-CoA is common to CHO, fat and protein metabolism eg.In citric acid cycle, cleavage of thioester in succinyl-CoA provides energy for synthesis of GTP Thioesters: high energy bond

Lipoamide cofactor in E 2 So… lipoamide swings to E3 to be reoxidised and transfer electrons to NADH via FAD now we have acetyl-CoA  Kreb’s, FA synthesis next must regenerate lipoamide and produce NADH Remember: there are multiple copies of each enzyme in complex So far…. disulfide swings to outer shell to collect hydroxyethyl from TPP in E1 swings to E2 to transfer acetyl to CoA

REDUCTION in E 3 OXIDATION in E 2 4. regeneration of lipoamide (E 2 ) by FAD (E 3 )

REDUCTION in E 2 OXIDATION in E 3 NAD + NADH + H + FAD funnels electrons to NAD +  NADH regeneration of FAD in E 3 5. redox

ENZYMECOFACTOR E1: pyruvate dehydrogenase +TPP E2: dihydrolipoyl transacetylase+ lipoamide E3: dihydrolipoyl dehydrogenase+ FAD

PDH controlled by covalent modification and product inhibition mammalian complex also contains kinase and phosphatase active E1 inactive E1 PDH kinase P PDH phosphatase Ser pyruvate AcCoA NAD + NADH CO 2 ATP

inhibit PDH high energy state active E1 inactive E1 PDH kinase P PDH phosphatase Ser pyruvate AcCoA NAD + NADH CO 2 activates ATP

inhibition by products in addition to activating PDH kinase, NADH and acetyl-CoA: compete with substrates for binding sites drive E2 and E3 in reverse (these reactions are close to equilibrium) E2 not available to collect hydyrxyol from TPP TPP cannot accept pyruvate

activate PDH low cell energy, or high available fuel active E1 inactive E1 PDH kinase P PDH phosphatase Ser pyruvate AcCoA NAD + NADH CO 2 glucose  Insulin activates ADP CoA

activate PDH pyruvate overrides NADH, AcCoA  still make AcCoA for fat when  pyruvate active E1 inactive E1 PDH kinase P PDH phosphatase Ser pyruvate AcCoA NAD + NADH CO 2 CoA activates

pyruvate AcCoA glucose citric acid cycle OAA malonyl- CoA PDH AC Carbox gluconeogenesis fatty acids Pyr carbox in high energy: (high ATP, high AcCoA, high NADH)  gluconeogenesis, fatty acid synthesis in low energy (low ATP, low AcCoA, )  glycolysis FA Synthase PEP PK CO 2 We now look at 3 other enzymes that use ‘swinging arm’ cofactors Pyruvate carboxylase AcetylCoA carboxylase Fatty acid synthase

pyruvate carboxylase first reaction in gluconeogenesis with PEPCK to bypass pyruvate kinase (  G<<0 in glycolysis) requires ATP to overcome –ve  G o ‘ of glycolysis + HCO 3 - pyruvate (3C) oxaloacetate (4C) ATPADP glucose (6C) another good animation, if you can access it: (not examinable, but might help understanding!)

tetramer each monomer has 2 active sites uses biotin as swinging arm pyruvate carboxylase

biotin HCO 3 - biotin’s swinging arm carboxyphosphate ATP ADP carboxybiotin in active site 1 Biotin carboxylation is catalyzed at one active site : first, ATP reacts with HCO3- (bicarbonate) to yield carboxyphosphate. The carboxyl from this high energy phosphate intermediate is transferred to the nucleophilic N of the biotin ring

At active site 1: 1. bicarb + ATP  high energy carboxyphosphate intermediate  2. -ve  G transfer of CO 2 to biotin = carboxylation I won’t ask you to recreate bond rearrangements!

biotin HCO 3 - pyruvate (3C) oxaloacetate (4C) carboxyphosphate ATP ADP carboxybiotin 2. biotin arm swings to the 2nd active site, active CO 2 is transferred from carboxybiotin to pyruvate  OAA

at active site 2: 1. CO 2 leaves biotin,2. biotin accepts a proton from pyruvate 3. pyruvate attacks CO 2 OAA nucleophile (donates e - ) I won’t ask you to recreate bond rearrangements! pyruvate loses a proton, becomes an enolate

biotin HCO 3 - biotin’s swinging arm pyruvate (3C) oxaloacetate (4C) carboxyphosphate ATP ADP carboxybiotin Overall: at active site 1: biotin + ATP + HCO3-  carboxybiotin + ADP + Pi at active site 2: carboxybiotin + pyruvate  OAA + biotin

AcetylCoA carboxylase first reaction committed step in fatty acid synthesis Also uses biotin as swinging arm between two active sites reactions very similar to pyruvate carboxylase + HCO 3 - AcetylCoA (2C) malonylCoA (4C) ATPADP fatty acids

biotin HCO 3 - biotin’s swinging arm Acetyl-CoA (2C) malony-lCoA (3C) carboxyphosphate ATP ADP carboxybiotin WOW look! mechanism of carboxylation (addition of COO-) is the same as for pyruvate carboxylase!!! ATP-dependent carboxylation of the biotin, carried out at active site 1, is followed by transfer of the carboxyl group to acetyl-CoA at a second active site 2. only difference is COO- is added to acetylCoA rather than to pyruvate

regulation of AcCoA-Carboxylase The mammalian enzyme is regulated, by  phosphorylation by cAMP dependent kinase  inhibition when  energy (  cAMP)  allosteric control by local metabolites. Conformational changes with regulation:  active = multimeric filamentous complexes.  inactive = dissociation to = monomeric form P

fatty acid synthase dimer 6 active sites are individual domains of a large protein –? developed from gene fusion –has more catalytic activities than any enzyme! has two prosthetic groups  thioester bonds –thiol of cysteine (in condensing domain) –thiol of P-pantetheine (in acyl carrier domain) acts as a long flexible arm transferring substrates between active sites

has two prosthetic groups Phosphopantetheine is covalently linked to a serine of the acyl carrier protein domain The long flexible arm of phosphopantetheine allows its thiol to move between active sites forms thioesters like CoA does thiol of cysteine in condensing domain thiol of P-pantetheine

phosphopantetheine is part of CoA

fatty acid synthase 2)Thioester bond between malonyl and pantetheine 3)The condensation reaction * involves decarboxylation of the malonyl  carbanion  attacks carbonyl carbon of the acetyl. Uses swinging arm of pantotheine You will have done these reactions in Dr Denyer’s lectures 2NADPH H20H20 3 2

dimer of the multi-domain enzyme are probably aligned in antiparallel In the transfer step: the growing fatty acid chain is preferentially passed from the pantetheine thiol of one subunit  cysteine thiol of the other ? intra-subunit substrate transfers also occur by swinging arm of pantetheine

essential dietary cofactors: cannot be made by mammals thiamine = vitamin B1 (in TPP) –deficiency = beri-beri –eg alcohol  reduced uptakeof thiamine  brain symptoms (  brain glucose metabolism) riboflavin = vitamin B2 (for FAD) niacin = vitamin B3 (NAD) lipoic acid biotin pantothenic acid (vitamin B5)

advantages of multi-active site enzymes and multi enzyme complexes  diffusion distance between substrate and active sites (usually the limiting factor in determining the reaction rate)  reaction rate  chance of side reactions –substrates stay within complex coordinated control of sequential reactions

Voet, Voet and Pratt (2nd Ed).  G and equilibrium pg 401 PDH pg , regulation pg 533 TPP mechanism pg 450 thioester bonds pg 413 Pyruvate carboxylase pg 502, pg AcetylCoA carboxylase pg 651 Fatty acid synthase pg 653 (much more detail than you need for this lecture!)