INTEGRATION OF METABOLISM

Metabolism consists of catabolism and anabolism

Catabolism: degradative pathways– Usually energy-yielding!

Anabolism: biosynthetic pathways– energy-requiring!

Catabolism and Anabolism

Catabolic pathways converge to a few end products

Anabolic pathways diverge to synthesize many biomolecules

Some pathways serve both in catabolism and anabolism

Such pathways are amphibolic

Organization in Pathways

Pathways consist of sequential stepsThe enzymes may be separateOr may form a multienzyme

complexOr may be a membrane-bound

systemNew research indicates that

multienzyme complexes are more common than once thought

Mutienzyme complex

Separateenzymes

Membrane Bound System

Organization of Pathways

Linear(product of rxns are

substrates for subsequent rxns)

Closed Loop(intermediates recycled)

Spiral(same set of enzymes

used repeatedly)

Themes in Metabolic Regulation

• Allosteric regulation

• Covalent modification

• Control of enzyme levels

• Compartmentalization

• Metabolic specialization of organs

Allosteric Regulation

End products are often inhibitors

Allosteric modulators bind to site other than the active site

Allosteric enzymes usually have 4o structure

Vo vs [S] plots give sigmoidal curve for at least one substrate

Can remove allosteric site without effecting enzymatic action

Regulation of Enzyme Activity(biochemical regulation)

1st committed step of a biosynthetic pathway or enzymes at pathway branch points often regulated by feedback inhibition.

Efficient use of biosynthetic precursors and energy

B A C1 3”

3’

2

E F G4’ 5’

H I J4” 5”

XX

Vo vs [S] plots give sigmoidal curve for at least one substrate

Binding of allosteric inhibitor or activator does not effect Vmax, but does alter Km

Allosteric enzyme do not follow M-M kinetics

Pengaruh aktivator ADP terhadap aktivitas enzim Fosfofruktokinase

(FPK1)

Allosteric T to R transition

Concerted model Sequential model

ET-I ET ER ER-SI

I S

S

Allosteric modulators bind to site other than the active site and allosteric enzymes have

4o structure

Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP

ADP

Allosteric Activator (ADP) binds distal to active site

Regulation of Hexose Transporters

Intra-cellular [glucose] are much lower than blood [glucose].

Glucose imported into cells through a passive glucose transporter.

Elevated blood glucose and insulin levels leads to increased number of glucose transporters in muscle and adipose cell plasma membranes.

Covalent Modification

• Covalent modification of last step in signal transduction pathway

• Allows pathway to be rapidly up or down regulated by small amounts of triggering signal (HORMONES)

• Last longer than do allosteric regulation (seconds to minutes)

• Functions at whole body level

Covalent modification

•Regulation by covalent modification is

slower than allosteric regulation•Reversible•Require one enzyme for activation and one

enzyme for inactivation•Covalent modification freezes enzyme T or

R-conformation

Phosphorylation/dephosphorylation

•Most common covalent modification

•Involve protein kinases/phosphatase

•PDK inactivated by phosphorylation

•Amino acids with –OH groups are

targets for phosphorylation

•Phosphates are bulky (-) charged

groups which effect conformation

Enzyme Levels• Amount of enzyme determines rates

of activity

• Regulation occurs at the level of gene expression

• Transcription, translation

• mRNA turnover, protein turnover

• Can also occur in response to hormones

• Longer term type of regulation

Regulation of Gene Expression

AAAAAA5’CAPmRNA

RNA Processing

RNA Degradation

Protein Degradation

Post-translational modification

Activeenzyme

Compartmentalization

One way to allow reciprocal regulation of catabolic and anabolic processes

Metabolic Specialization of Organs

Specialization of Organs

• Regulation in higher eukaryotes

• Organs have different metabolic rolesi.e. Liver = gluconeogenesis,

Muscle = glycolysis

• Metabolic specialization is the result of differential gene expression

Brain• Glucose is the primary fuel for the brain

• Brain lacks fuel stores, requires constant supply of glucose

• Consumes 60% of whole body glucose in resting state. Required too maintain Na and K membrane potential in of nerve cells

• Fats can’t serve as fuel because blood brain barrier prevents albumin access.

• Under starvation can ketone bodies used.

Muscle• Glucose, fatty acids and ketone bodies are

fuels for muscles

• Muscles have large stores of glycogen (3/4 of body glycogen in muscle)

• Muscles do not export glucose (no glucose-6-phosphatase)

• In active muscle glycolysis exceeds citric acid cycle, therefore lactic acid formation occurs

• Cori Cycle required

Siklus Cori (Cori Cycle)

Muscle• Muscles can’t do urea cycle. So

excrete large amounts of alanine to get rid of ammonia (Glucose Alanine Cycle)

• Resting muscle uses fatty acids to meet 85% of energy needs

Heart Muscle

• Heart exclusively aerobic and has no glycogen stores.

• Fatty acids are the hearts primary fuel source. Can also use ketone bodies. Doesn’t like glucose

Liver

• Major function is to provide fuel for the brain, muscle and other tissues

• Metabolic hub of the body

• Most compounds absorb from diet must first pass through the liver, which regulates blood levels of metabolites

Liver: carbohydrate metabolism

• Liver removes 2/3 of glucose from the blood

• Glucose is converted to glucose-6-phosphate (glucokinase)

• Liver does not use glucose as a fuel. Only as a source of carbon skeletons for biosynthetic processes.

• Glucose-6-phosphate goes to glycogen (liver stores ¼ body glycogen)

Liver: lipid metabolism• Excess glucose-6-phosphate goes to

glycolysis to form acetyl-CoA

• Acetyl-CoA goes to form lipids (fatty acids cholesterol)

• Glucose-6-phosphate also goes to PPP to generate NADH for lipid biosynthesis

• When fuels are abundant triacylglycerol and cholesterol are secreted to the blood stream in LDLs. LDLs transfer fats and cholesterol to adipose tissue.

• Liver can not use ketone bodies for fuel.

Liver: protein/amino acid metabolism

• Liver absorbs the majority of dietary amino acids.

• These amino acids are primarily used for protein synthesis

• When extra amino acids are present the liver or obtained from the glucose alanine cycle amino acids are catabolized

• Carbon skeletons from amino acids directed towards gluconeogenesis for livers fuel source

Adipose Tissue

Enormous stores of TriacyglycerolFatty acids imported into adipocytes

from chylomicrons and VLDLs as free fatty acids

Once in the cell they are esterified to glycerol backbone.

Glucagon/epinephrine stimulate reverse process

Well-Fed State

• Glucose and amino acids enter blood stream, triacylglycerol packed into chylomicrons

• Insulin is secreted, stimulates storage of fuels

• Stimulates glycogen synthesis in liver and muscles

• Stimulates glycolysis in liver which generates acetyl-CoA for fatty acid synthesis

Refed State

• Liver initially does not absorb glucose, lets glucose go to peripheral tissues, and stays in gluconeogenesis mode

• Newly synthesized glucose goes to replenish glycogen stores

• As blood glucose levels rise, liver completes replenishment of glycogen stores.

• Excess glucose goes to fat production.

Starvation

Fuels change from glucose to fatty acids to ketone bodies

GLIKOLISIS Glikolisis terdiri dari 2 fase: Fase preparasi (preparatory phase),

yaitu fosforilasi glukosa dan konversinya menjadi gliseraldehid 3-fosfat.

Fase pembayaran (payoff phase), yaitu

konversi oksidatif gliseraldehid 3-P menjadi piruvat disertai pembentukan ATP dan NADH.

Preparatory phase

Payoff phase

• Seven steps of glycolysis are retained

• Three steps are replaced

• The new reactions provide for a spontaneous pathway (G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

Control Points in Glycolysis

Regulation of Hexokinase

Glucose-6-phosphate is an allosteric inhibitor of hexokinase.

Levels of glucose-6-phosphate increase when down stream steps are inhibited.

This coordinates the regulation of hexokinase with other regulatory enzymes in glycolysis.

Hexokinase is not necessary the first regulatory step inhibited.

Regulation of PhosphoFructokinase (PFK-1)

PKF-1 has quaternary structureInhibited by ATP and CitrateActivated by AMP and Fructose-2,6-

bisphosphateRegulation related to energy status of

cell.

Effect of ATP on PFK-1 Activity

Effect of ADP and AMP on PFK-1 Activity

Regulation of fructose 1,6-bisphosphatase-1 (FBPase-1)

and phosphofructokinase-1 (PFK-1)

Regulation of PFK by Fructose-2,6-bisphosphate

• Fructose-2,6-bisphosphate is an allosteric activator of PFK in

eukaryotes, but not prokaryotes•Formed from fructose-6-phosphate by PFK-2•Degraded to fructose-6-phosphate by fructrose 2,6-

bisphosphatase.•In mammals the 2 activities are on the same enzyme•PFK-2 inhibited by Pi and stimulated by citrate

Regulation of Pyruvate Kinase

Allosteric enzymeActivated by Fructose-1,6-bisphosphate

(example of feed-forward regulation)Inhibited by ATPWhen high fructose 1,6-bisphosphate

present plot of [S] vs Vo goes from sigmoidal to hyperbolic.

Increasing ATP concentration increases Km for PEP.

In liver, PK also regulated by glucagon. Protein kinase A phosphorylates PK and decreases PK acitivty.

Pyruvate Kinase Regulation

Deregulation of Glycolysis in Cancer Cells

Glucose uptake and glycolysis is ten times faster in solid tumors than in non-cancerous tissues.

Tumor cells initally lack connection to blood supply so limited oxygen supply

Tumor cells have fewer mitochondrial, depend more on glycolysis for ATP

Increase levels of glycolytic enzymes in tumors (oncogene Ras and tumor suppressor gene p53 involved)

Gluconeogenesis

• Synthesis of "new glucose" from common metabolites

• Humans consume 160 g of glucose per day

• 75% of that is in the brain • Body fluids contain only 20 g of

glucose • Glycogen stores yield 180-200 g of

glucose • The body must still be able to make its

own glucose

Gluconeogenesis

• Occurs mainly in liver and kidneys

• Not the mere reversal of glycolysis for 2 reasons:– Energetics must change to make

gluconeogenesis favorable (delta G of glycolysis = -74 kJ/mol

– Reciprocal regulation must turn one on and the other off - this requires something new!

• Seven steps of glycolysis are retained

• Three steps are replaced

• The new reactions provide for a spontaneous pathway (G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

Regulation of Gluconeogenesis

• Reciprocal control with glycolysis • When glycolysis is turned on,

gluconeogenesis should be turned off • When energy status of cell is high,

glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose

• When energy status is low, glucose should be rapidly degraded to provide energy

• The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!

Transaminasi

The first of the bypass reactions in gluconeogenesis is the conversion of pyruvate to Phosphoenolpyruvate (PEP)

Conversion of Pyruvate to Phosphoenolpyruvate Requires Two Exergonic Reactions

Alternative paths from pyruvate to phosphoenolpyruvate

Pyruvate Carboxylase • The reaction requires ATP and bicarbonate as

substrates • Biotin cofactor• Acetyl-CoA is an allosteric activator • Regulation: when ATP or acetyl-CoA are high,

pyruvate enters gluconeogenesis

PEP Carboxykinase • Lots of energy needed to drive this

reaction!

• Energy is provided in 2 ways:

– Decarboxylation is a favorable reaction

– GTP is hydrolyzed

• GTP used here is equivalent to an ATP

PEP Carboxykinase Not an allosteric enzymeRxn reversible in vitro but irreversible

in vivoActivity is mainly regulated by control

of enzyme levels by modulation of gene expression

Glucagon induces increased PEP carboxykinase gene expression

Fructose-1,6-bisphosphatase

• Thermodynamically favorable - G in liver is -8.6 kJ/mol

• Allosteric regulation:– citrate stimulates – fructose-2,6--bisphosphate inhibits– AMP inhibits

Glucose-6-Phosphatase • Presence of G-6-Pase in ER of liver and kidney cells makes

gluconeogenesis possible • Muscle and brain do not do gluconeogenesis • G-6-P is hydrolyzed as it passes into the ER • ER vesicles filled with glucose diffuse to the plasma

membrane, fuse with it and open, releasing glucose into the bloodstream.

Regulation of Gluconeogenesis

• Reciprocal control with glycolysis • When glycolysis is turned on,

gluconeogenesis should be turned off • When energy status of cell is high, glycolysis

should be off and pyruvate, etc., should be used for synthesis and storage of glucose

• When energy status is low, glucose should be rapidly degraded to provide energy

• The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!

•Metabolites other than pyruvate can enter gluconeogenesis

•Lactate (Cori Cycle) transported to liver for gluconeogenesis

•Glycerol from Triacylglycerol catabolism

•Pyruvate and OAA from amino acids (transamination rxns)

•Malate from glycoxylate cycle -> OAA -> gluconeogenesis

The Metabolism of Glycogen in Animals

Glycogen granules in a hepatocyte

Hormonal Regulation of Glycogen Metabolism

InsulinSecreted by pancreas under high blood [glucose]Stimulates Glycogen synthesis in liverIncreases glucose transport into muscles and adipose

tissuesGlucagonSecreted by pancreas in response to low blood

[glucose]Stimulates glycogen breakdownActs primarily in liverEphinephrineSecrete by adrenal gland (“fight or flight” response)Stimulates glycogen breakdown. Increases rates of glycolysis in muscles and release of

glucose from the liver

Metabolism of Tissue Glycogen

• But tissue glycogen is an important energy reservoir - its breakdown is carefully controlled

• Glycogen consists of "granules" of high MW

• Glycogen phosphorylase cleaves glucose from the nonreducing ends of glycogen molecules

• This is a phosphorolysis, not a hydrolysis

• Metabolic advantage: product is a sugar-P - a "sort-of" glycolysis substrate

•Glycogen phosphorylase cleaves glycogen at non-reducing end to generate glucose-1-phosphate•Debranching of limit dextrin occurs in two steps.•1st, 3 X 1,4 linked glucose residues are transferred to non-reducing end of glycogen•2nd, amylo-1,6-glucosidase cleaves 1,6 linked glucose residue.•Glucose-1-phosphate is converted to glucose-6-phosphate by phosphoglucomutase

The catabolic pathways: from glycogen to glucose 6-phosphate

(glycogenolysis) and from glucose 6-phosphate to pyruvate

(glycolysis)The anabolic pathways: from pyruvate to glucose (gluconeogenesis)

and from glucose to glycogen (glycogenesis)

Glycogen Breakdown Is Catalyzed by Glycogen Phosphorylase

Glycogen Synthase • Forms -(1 4) glycosidic bonds in glycogen • Glycogen synthesis depends on sugar nucleotides UDP-

Glucose• Glycogenin (a protein!) protein scaffold on which glycogen

molecule is built.• Glycogen Synthase requires 4 to 8 glucose primer on

Glycogenin (glycogenein catalyzes primer formation)• First glucose is linked to a tyrosine -OH • Glycogen synthase transfers glucosyl units from UDP-glucose

to C-4 hydroxyl at a nonreducing end of a glycogen strand.

Branch synthesis in glycogen: The glycogen-branching enzyme (also called amylo (1→4) to (1→6) transglycosylase or glycosyl-(4→6)-transferase)

Coordinated Regulation of GlycogenSynthesis and Breakdown

Glycogen Phosphorylase Is Regulated Allosterically and Hormonally

Glycogen Synthase Is Also Regulated by Phosphorylation and Dephosphorylation

Control of Glycogen Metabolism

• A highly regulated process, involving reciprocal control of glycogen phosphorylase (GP) and glycogen synthase (GS)

• GP allosterically activated by AMP and inhibited by ATP, glucose-6-P and caffeine

• GS is stimulated by glucose-6-P

• Both enzymes are regulated by covalent modification - phosphorylation

Glycogen phosphorylase of liver as a glucose sensor

Hormonal Regulation of Glycogen Metabolism

Effect of glucagon and epinephrine on glycogen phosphorylase glycogen

synthase activities

Effect of insulin on glycogen phosphorylase and glycogen

synthase activities

LIPOPROTEIN

Processing of dietary lipids in vertebrates

Lipoproteins

Transport water insoluble TAG, cholesterol and cholesterol-esters throughout circulatory system

Hydrophobic core containing TAG and cholesterol-esters

Hydrophillic surface made of proteins (apoproteins) and phospholipids)

Common membrane phospholipids

P

O

OO

O

H

CH2HCH2C

O O

C C OO

R1 R2

P

O

OO

O

CH2

CH2HCH2C

O O

C C OO

R1 R2

CH2

NH3

P

O

OO

O

CH2

CH2HCH2C

O O

C C OO

R1 R2

CH2

NH3

COO

P

O

OO

O

CH2

CH2HCH2C

O O

C C OO

R1 R2

CH2

NH3C CH3

CH3

Phosphatidate Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine

Fatty acids and MAG enter mucosal cells where they are used to re-synthesize TAG

TAG is then packaged into lipoprotein transport particles called chylomicrons (lipoprotein).

Chylomicrons are mainly composed of TAG and apoprotein B-48. Also contain fat solubel vitamins

Chylomicrons enter the lymph system and then the blood stream.

Chylomicrons bind to membrane bound lipoprotein lipases at the surface of adipose and muscle cells.

Lipoproteins

Several different classes of lipoproteins.Chylomicrons deliver dietary fats to tissuesVLDL, IDL and LDL transport endogenously

synthesized TAG and cholesterol to tissuesHDLs remove cholesterol from serum and

tissues and transports it back to the liver.VLDL, IDL, LDL, and HDL named based on their

density. Low density lipoproteins have high lipid to protein ratios. High density lipoproteins have low lipid to protein ratios.

Lipoproteins

Lipases in capillaries of adipose and muscle tissues degrade TAG in VLDLs. VLDLs become IDLs.

IDLs can then give up more lipid and become LDLs.

LDLs are rich in cholesterol and cholesterol-esters.

Apolipoproteins

VLDLs, IDLs, and LDLs all contain a large monomeric protein called ApoB-100.

ApoB-100 forms amphipathic crust on lipoprotein surface.

Chylomicrons contain analogous lipoprotein ApoB-48.

VLDLs and IDLs also possess a number of small weakly associated proteins that disassociate during lipoprotein degradation.

Small apolipoproteins function to modulate the activity of enzymes involved in lipid mobilization and interactions with cell surface receptors.

LDL Receptor

Binds to ApoB-100.Found on cell surface of many cell typesMediates delivery of cholesterol by inducing

endocytosis and fusion with lysosomes.Lysosomal lipases and proteases degrade

the LDL. Cholesterol then incorporates into cell membranes or is stored as cholesterol-esters.

High LDL levels can lead to cardiovascular disease.

LDL can be oxidized to form oxLDLoxLDL is taken up by immune cells called

macrophages.Macrophages become engorged to form foam

cells.Foam cells become trapped in the walls of

blood vessels and contribute to the formation of atherosclerotic plaques.

Causes narrowing of the arteries which can lead to heart attacks.

Plaque Build up in Artery

Absence of LDL Receptor Leads to Hypercholesteremia and Atherosclerosis

Persons lacking the LDL receptor suffer from familial hypercholesterolemia

Result of a mutation in a single autosomal gene

Total plasma cholesterol and LDL levels are elevated.

Homozygous individuals have cholesterol levels of 680 mg/dL. Heterozygous individuals = 300 mg/dL. Healthy person = <200 mg/dL.

Most homozygous individuals die of cardiovascular disease in childhood.

LDLs/HDLs and Cardiovascular Disease

LDL/HDL ratios are used as a diagnostic tool for signs of cardiovascular disease

LDL = “Bad Cholesterol”HDL = “Good Cholesterol”A good LDL/HDL ratio is 3.5Protective role of HDL not clear.An esterase that breaks down oxidized lipids

is associated with HDL. It is possible (but not proven) that this enzyme helps destroy oxLDL