cellular metabolism

1. Cellular Metabolism and Metabolic Disorders
1. Cellular Metabolism
Metabolism, the sum of all the chemical
transformations taking place in a cell or organism,
occurs through a series of enzyme-catalyzed
reactions.
Each of the consecutive steps in a metabolic
pathway brings about a specific, small chemical
change, usually the removal, transfer, or addition
of a particular atom or functional group.
precursor metabolites product

2. Metabolism is the overall process through which living systems acquire and
utilize the free energy they need to carry out their various functions.
 It is called amphibolic -having both catabolism and anabolism pathway
 Metabolism consists of catabolism (oxidative) and anabolism (reductive)
1. Catabolism: Energy-yielding degradative metabolic pathways (Exergonic)
E.g. Cellular Respiration
2. Anabolism: Energy-requiring biosynthetic metabolic pathways (Endergonic)
E.g. photosynthesis
 Metabolic Pathways consist of sequential steps of reaction(Closed Loop-
intermediates recycled or Linear -product of reaction are substrates for
subsequent reaction)
 They do so by coupling the exergonic reactions of nutrient oxidation to the
endergonic processes required to maintain the living state such as:
√ the performance of mechanical work,
√ the active transport of molecules against concentration gradients
√ the biosynthesis of complex molecules.

3. 2
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4. Enzymes and their role in metabolism
Enzymes: are protein biocatalysts that increase the rate of reaction
without being change themselves in the overall process.
Biocatalysts: are “Biological protein catalysts" that Speed up reactions
in the body of an organism
All metabolic reaction in the body are mediated by enzymes
Enzymes lower the activation energy required for a reaction to occur
Enzymes are protein catalysts that speed biochemical reactions by
facilitating the molecular rearrangements that support cell function.
All known enzymes are proteins with the exception of recently
discovered RNA enzymes. Some enzymes may additionally contain a
non-protein group.

5. Classification of enzymes
 Many enzymes require the presence of other compounds (cofactors)
before their catalytic activity can be exerted. This entire active
complex is referred to as the holoenzyme (apoenzyme (protein
portion) plus the cofactor (coenzyme, prosthetic group or metal-ion
activator)).
 on the basis of differences in chemical nature, the enzymes may be
described as :
1. Simple enzymes: Simple enzymes are made up of only protein
(polypeptide). They contain no chemical groups other than amino acid
residues. Digestive enzymes such as pepsin and trypsin are of this
nature.
2. Conjugate Enzymes: It is an enzyme which is formed of two parts – a
protein part called apoenzyme (e.g., flavoprotein) and a non-protein
part called cofactor(organic or inorganic).
3. Metallo-enzymes: The metal cofactors involved in enzymic reactions
are monovalent (K+ ) and divalent cations (Mg++, Mn++ and Cu++).
4. Isoenzymes (Isozymes): (multiple forms of enzymes) are enzymes that
differ in amino acid sequence but catalyze the same chemical
reaction

6.  Classes of enzymes based on the substrate they act up on or
according to their catalytic function enzymes are grouped into six
1.Oxidoreductases: Catalyze an oxidation-reduction reaction
between two substrates.
2.Transferases: Catalyze the transfer of a group other than
hydrogen from one substrate to another.
3. Hydrolases: Catalyze hydrolysis of various bonds.
4. Lyases: Catalyze removal of groups from substrates without
hydrolysis. The product contains double bonds
5.Isomerases: Catalyze the interconversion of geometric,
optical, or positional isomers.
6.Ligases: Catalyze the joining of two substrate molecules,
coupled with breaking of the pyrophosphate bond in
adenosine triphosphates (ATP) or a similar compound.

7. Mechanism of Enzyme Action
► There are two proposed models to explain the specificity of the
interaction between the substrate molecule and the active site of
an enzyme.
Lock And Key Model: fixed (stationary) model (theory) of
enzyme action. the lock is analogs to the enzyme and the key is
the substrate.
 Only the correctly sized key (substrate) fits into the key hole
(active site) of the lock (enzyme).
Induced-fit Model: The induced-fit model of enzyme
action assumes that the enzyme conformation changes to
accommodate the substrate molecule. it is a
dynamic(flexible) model of enzyme action.
 Is a model of enzyme activity that describes an enzyme
as a dynamic protein molecule that changes shape to
better accommodate the substrate.

8. The basic mechanism by which enzymes catalyze chemical reactions begins
with the binding of the substrate (S ) to the active site of the enzyme (E ).
Substrate is the reactant(substance) that an enzyme acts on when it
catalyzes a chemical reaction.
Active Site is the specific region of the enzyme which combines
with the substrate or it is the location where the substrate binds to
an enzyme
The attachment (binding) of the substrate (s) to the active site of an
enzyme (E) creates the enzyme–substrate complex (ES ).
That is E + S  ES
The binding of the substrate to the enzyme causes changes in the distribution of
electrons in the chemical bonds of the substrate and ultimately causes the
reactions that lead to the formation of products (P). That is ES  E + P
The products are released from the enzyme surface to regenerate the enzyme for
another reaction cycle. The overall reaction for the conversion of substrate to
product can be written as : E + S  ES  E + P

9. Factors affecting enzymatic activities
 The ability of an enzyme to bind to its substrate and effectively catalyze a reaction is
largely determined by its three dimensional structure and certain environmental
conditions.
 What are the environmental conditions that optimize enzyme activity and how
do changes affect enzyme function?
1.Temperature:
 Enzyme catalyzed reactions increase in speed (rate or velocity) with an
increase in temperature.
 However, as the temperature increases beyond a critical point
(optimum), thermal agitation begins to disrupt protein structure,
resulting in Denaturation of enzyme and loss of enzyme function.
 Every enzyme has an optimal temperature at which it works best.
Enzyme activity decreases above and below the optimal temperature.
 Most human enzymes work best at around 37°C, normal body
temperature and Peaks at ~ 37 - 40°C then drops rapidly b/c of
denaturation of the enzyme(protiens)
 If the temperature is below critical point, enzymes are being
inactive (not functional)

10. Enzyme denature
at high temperature
so reaction rate fail
(decrease rapidly )
Optimum
temperature range
from 37oC-40oC

11. 2. pH (acidic or alkaline ):
 Enzymes function within an optimal pH range. Each enzyme exhibits
peak activity at narrow pH range (pH optimum).
 If pH changed, so is no longer within the enzyme range; reaction will
decrease.
 pH optimum reflects the pH of the body fluid (solution) in which the
enzyme is found (the enzyme’s working environment).
 Every Enzymes have their own optimal pH in which they work best.
 Fore example, the digestive enzyme pepsin works best in the acidic
environment of the stomach (pH =2) whereas
 The digestive enzyme trypsin has an optimal pH of 8 and works best in
the alkaline environment of the small intestine.
 Enzymes are most active at optimum pH. Amino acids with acidic or
basic side-chains have the proper charges when the pH is optimum.
 Enzyme Activity is lost at low or high pH as tertiary structure is
disrupted
 Usually most enzymes have the optimum pH between 6 and 8

12. Most Enzymes function best at Optimum pH (pH
Between 6 and 8)

13. 3. Substrate Concentration:
 Enzyme catalyzed reaction rate increases as the substrate
concentration increases up to a point of saturation.
 However, beyond Enzyme-catalyzed reactions can be saturated,
increasing the substrate concentration can not increase reaction
rate of an enzyme
 There are a limited number of specific enzyme molecules in a cell
at any one time.
 Since it takes some time for a catalyst to catalyze a particular
reaction, the speed at which a catalyzed reaction proceeds cannot
increase indefinitely by increasing the concentration of the
substrate.
 The limiting factor in the reaction may be the amount of substrate
or the amount of enzyme available
 At a specific [enzyme], rate of product formation increases as the
[substrate] increases.

14.  Plateau of maximum velocity occurs when enzyme is saturated. At this
point adding an extra Additional [substrate] does not increase reaction
rate
 The rate (velocity) of a reaction is being remain constant at enzyme-
substrate saturation point when all enzyme occupied by substrates
 The rate of reaction increases as substrate concentration increases (at
constant enzyme concentration)
 Maximum activity occurs when the enzyme is saturated (when all
enzymes are binding substrate)
 The relationship between reaction rate and substrate concentration is
exponential, and asymptotes (levels off) when the enzyme is saturated

15. (Saturation)

16. 4. Enzyme Concentration:
 The rate of reaction increases as enzyme concentration increases
(at constant substrate concentration)
 At higher enzyme concentrations, more enzymes are available to
catalyze the reaction (more reactions at once)
 There is a linear relationship between reaction rate and enzyme
concentration (at constant substrate concentration)

18. 5. Inhibitors (I):
 are molecules that cause a loss of enzyme catalytic activity.
 They prevent substrates from fitting into the active site of the
enzyme.
 A variety of substances inhibit an enzyme catalytic activity.
 Enzyme inhibitors are among substances that diminish enzyme
catalytic activity and decrease reaction rate
6. Effectors: are molecules or substances that increase the
catalytic activity of an enzyme and increase reaction rate

19. Types of enzyme Inhibitors
Enzyme Inhibitors: are low molecular weight chemical compounds that can
reduce or completely inhibit the enzyme catalytic activity either reversibly or
permanently (irreversibly). Enzyme inhibitors are molecules that reduce the
catalytic activity of enzymes. Reducing of effective enzymatic activity or
complete blocking of enzyme may cause either complete death of cells or
modification in the pathways.
There are two types of Enzyme inhibitors based on their reversibility. These are
1. Reversible inhibitors:
 Goes on and off, allowing the enzyme to regain activity when the inhibitor
leaves
 Bind to enzyme with only non-covalent bond temporarily
 Do not perform any chemical change in enzyme or themselves
 Can be removed from enzyme completely by reducing inhibitor concentration

20. 2. Irreversible inhibitors:
 Bind to enzyme with covalent bond and prevent the enzyme from further
performing of catalytic activity
 perform a chemical change in enzyme (destroy the active center of the enzyme
structure)
 Cannot be removed from enzyme
 destroys enzyme activity, usually by bonding with side-chain groups in the active
site
 Inhibitors are covalently bound to the essential groups of enzymes.
 Inhibitors cannot be removed with simple dialysis or super-filtration.
 Binding can cause a partial loss or complete loss of the enzymatic activity.
 The three common types of reversible inhibitions are:
►Competitive reversible inhibition.
►Uncompetitive reversible inhibition.
►non-competitive inhibition

21. Competitive Reversible Inhibitors:
 are reversible inhibitors that bind to the active site of an enzyme
 compete with the normal substrate for an enzyme’s active site
 Are so structurally similar to the enzyme’s substrate that they are able
to enter the enzyme’s active site and block the normal substrate from
binding with active site of an enzyme
 Their binding process with an enzyme is Reversible and can be
overcome by increasing the concentration of the enzyme’s substrate,
allowing it to compete favorably with the inhibitor.
 its effect is reversed by increasing substrate concentration
 Inhibition depends on the affinity of enzymes and the ratio of [E] to [S].
 As [S] increases, the effect of inhibitors is reduced, leading to no change
in Vmax

22. Non-competitive Reversible Inhibitors:
 Are reversible inhibitors that bind to an allosteric site (not the active
site) of an enzyme and cause a conformational change in the enzyme
(distorts the shape of the enzyme), preventing the normal substrate from
binding to the active site of an enzyme and lead to the loss of enzyme
activity
 can bind to both free enzyme and the ES complex
 Do not have a structural similarity with an enzyme’s substrate
 Do not compete with an enzyme’s substrate for the active site.
 The effect can not be overcome by increasing the concentration of the
enzyme’s substrate
 Instead they attach to another site on the enzyme and causing a change
in the enzyme’s shape. This process changes the active site in such a
way that it loses affinity for its substrate.

23.  Alternatively, the inhibitor may affect those parts of the
active site that perform the work of catalysis, resulting in a
loss of enzyme activity
 They are substances that attach to a binding site on an
enzyme other than the active site, causing a change in the
enzyme’s shape and a loss of affinity for its substrate
 The E-I complex formation does not affect the binding of
substrates.
 The e-I-S complexes do not proceed to form products.
 Reduce (decrease )Vmax and unchanged (do not affect ) Km.

24. Uncompetitive Reversible Inhibitors:
 Are reversible inhibitors that bind to the enzyme substrate complex but
not to the free enzyme
 The result is a decrease in reaction rate and the affinity of the enzyme to
substrate
 The effect of an uncompetitive inhibitor can not be overcome by high
concentrations of the substrate
 bind only to the enzyme-substrate complexes.
 The E-I-S complexes do not proceed to form products.
 The E-I-S complexes do not backward to the substrates and enzymes.
 This inhibition has the effects on reducing both Vmax and Km.
 Used Commonly in the multiple substrate reactions.
Allosteric sites: are receptor sites found some distance from the
active site of certain enzymes and used to bind substances that may
inhibit(inhibitors) or stimulate (activators) an enzyme’s activity

25. Cellular Respiration (Catabolism)
 Cells derive their energy from the chemical bonds of organic molecules.
Cellular respiration is the metabolic process of breaking down of
organic molecules aerobically (with oxygen) or anaerobically (without
oxygen) to release energy.
 The metabolic processes that break down sugars and other food sources
to derive energy consist of two major types due to the use of oxygen.
a. Anaerobic: Break down 6C sugars to 3C or 2C compounds to derive
some energy without oxygen. E.g. glycolysis, fermentation
b. Aerobic process: Break down 6C sugars in presence of oxygen to CO2
and H2O and provide most efficient source of energy.
E.g. pyruvate oxidation, TCA cycle and electron transport
chain(ETC)

26. There are two basic mechanism of ATP formation
in cellular respiration. These are:
Substrate-level Phosphorylation: The ATP that are made in
glycolysis come from transfer of phosphate groups from the
phosphorylated 3C sugars to ADP. Transfer of a phosphate
from a substrate to ADP directly is called “substrate-level
phosphorylation”.
Oxidative Phosphorylation: ATP production in the process
of cellular respiration mostly come from “oxidative
phosphorylation” in which the energy from electron
transport is conserved as a proton gradient that drives ATP
formation by ATP synthase.

27. There are four consecutive phases in cellular respiration.
1. Glycolysis
-occurs in the cytosol (cytoplasm ) without oxygen(does not require
O2) and it is called anaerobic respiration
3. Krebs (TCA) Cycle
-occurs in the mitochondrion matrix with oxygen (Requires oxygen)
- It is called aerobic respiration and C in glucose is released as CO2
-high-energy electrons produced via the molecule NADH and FADH2
4. Electron Transport Chain (ETC)
-occurs in the mitochondrion inner membrane with oxygen(Requires
oxygen) and It is called aerobic respiration
-most number of ATP’s produced
2.Pyruvate oxidation:is the Conversion of Pyruvate to Acetyl-CoA. It is aerobic
process that takes place in mitochondrial matrix. It is a link b/n glycolysis and TCA cycle.

28. 1. Glycolysis
 Glycolysis is an anaerobic process through which ATP is
synthesized during the conversion of the six-carbon sugar
glucose to two molecules of the three-carbon compound
pyruvate.
 Glycolysis means “splitting of sugar”.
 Glucose a 6C sugar is split into two 3C sugars and the 3C sugars
are further oxidized.
 Glycolysis is the first of the four phases of cellular respiration
 In glycolysis, glucose is broken down in the cytosol into two
molecules of pyruvate.
 The products of glycolysis include some stored energy as ATP,
NADH, which can be a source of additional energy and the 3
carbon compound pyruvate, which can be oxidized further by
the Krebs cycle.
 The catabolic pathway of glycolysis consists of ten steps each
catalyzed by a specific enzyme. We can divide these steps into
two phases:

29. a. Energy Investment Phase: requires two molecules of ATP (one
ATP at step 1 and one ATP at step 3) and Cleavage of 1 hexose to
2 triose
b. Energy Pay-off Phase:
 Makes ATP by “substrate-level phosphorylation” and makes
NADH by oxidation/reduction.
 Generate 2 ATPs, 2 NADH and 2 Pyruvates from a single
molecule of glucose
 involves two very high energy phosphate donating intermediates
or substrates in glycolysis. These are 1,3- bisphospho glycerate
(BPG) and Phosphoenolpyruvate (PEP)

31. The End Products (Net Results) of Glycolysis
 2 molecules of pyruvate(pyravic acid)
 2 ATP by substrate level phosphorylation
 2NADH and 2H+
Net Equation of glycolysis:
Glucose + 2NAD+ + 4e- + 4H+ → 2 Pyruvate + 2H20 +
2ATP + 2NADH + 2H+

32. The Three Fates of Pyruvate
Pyruvate  acetyl-CoA
– Occurs in mitochondria
– Produce CO2 and NADH + H+
– Catalyzed by Pyruvate Dehydrogenase(PDH)
– Aerobic **Acetyl-CoA used in the TCA cycle
Pyruvate  Lactate (occurred in muscles during rapid exercise)
– Produce CO2 and NAD+
– lactic acid fermentation and Anaerobic
– Pyruvic acid + NADH → lactic acid + Co2 + NAD+
– Catalyzed by Lactate dehydrogenase (LDH)
Pyruvate  Ethanol (occurred In yeast and few microorganisms)
– Produce CO2 and NAD+
– Alcoholic fermentation and Anaerobic
– Pyruvic acid + NADH → ethanol + Co2 + NAD+
– Catalyzed by alcohol dehyrogenase ( ADH)

33. Regulation of Glycolysis
 Because the principle function of glycolysis is to produce ATP, it
must be regulated so that ATP is generated only when needed
 Glycolysis is a tightly regulated pathway.
 The three irreversible steps of glycolysis are catalyzed by
hexokinase, phosphofructokinase and pyruvate kinase.
 All three of these enzymes are allosterically regulated.
 Only the enzymes found on the irreversible steps of glycolysis
(step-1,step-3 and step-10) (hexokinase, phosphofructokinase and
pyruvate kinase) can be regulated.

34.  Regulation of these enzymes can occur by several ways: that is
may regulated by :
 allosteric modulators (activators & inhibitors)
 hormone control (through phosphorylation/dephosphorylation)
 de novo synthesis
 Among these enzymes, Phosphofructokinase is by far the most
regulated enzyme.
 Because The enzyme which controls the flux of metabolites
through the glycolytic pathway is phosphofructokinase (PFK).
 PFK is an allosteric enzyme that occupies the key regulatory
position for glycolysis. This enzyme is regulated by.
 I. ATP feedback inhibition.
 II. AMP reverses inhibition.
 III Other allosteric effectors of PFK like ADP

35. 2. Pyruvate Oxidation
 The process of converting pyruvate into acetyl CoA is called
pyruvate oxidation. There is no ATP formation in pyruvate
oxidation but it used to generate acetyl CoA.
 The entry to the TCA cycle is through acetyl-CoA.
 Pyruvate is converted into acetyl-CoA by the enzyme pyruvate
dehydrogenase which is located inside the mitochondria.
 It is highly regulated step to properly regulate the TCA cycle
because this step is responsible for the availability of acetyl CoA
which is the raw material for TCA cycle

36. pyruvate dehydrogenase
The end products of pyruvate oxidation when 2pyruvate are:-
2 molecules of acetyl CoA,
2NADH and 2H+
2CO2

37. 3. Kerb Cycle (TCA)
 Much more energy can be obtained from glucose if it is oxidized
completely to CO2 and H2O through the TCA cycle and electron
transport system (requires O2 and mitochondria).
 In the presence of oxygen pyruvate, product of glycolysis, will enter into
the citric acid cycle and further oxidized to H2O and CO2.
 The citric acid cycle is also known as the tricarboxylic acid (TCA) cycle
and the Krebs cycle.
 In eukaryotes, the reactions of the citric acid cycle occur in the
mitochondria matrix
 Is aerobic respiration (it requires oxygen)
 Produce two molecules of ATP(2ATP) and six molecules of NADH
(6NADH) and two FADH2 which used by ETC by two turn of the Citric
acid cycle because we have 2 acetyl-CoA that enter to TCA cycle
 But 3 Molecules of NADH and 1 molecule of FADH2 are generated in
each turn of the Citric acid cycle.

38. The starting and ending substance for kerb cycle
is oxaloacetic acid (oxaloacetate)- the acetyl
CoA receptor/ accepter molecule in TCA cycle.
For a degradation of a single glucose there is a
formation of two acetyl CoA and so there is two
cycle (Turn) per glucose molecule
The citric acid cycle consists of eight main steps
The high energy phosphate donating
substrate/intermidate to ADP to syntesize ATP in
TCA cycle is called succinyl-CoA.
 The mechanism of ATP formation/production in TCA cycle is
done by substrate level phosphorylation.

40. End products of TCA cycle
 From the degradation of a single molecules of glucose 2
molecules of acetyl-CoA are enter the kerb cycle by two round
(one acetyl-coA at one round), then the cycle produces:
1) 6NADH (2NADH at step-3, 2NADH at step-4 and 2NADH at step-8)
2) 2FADH2 at step-6
3) 4 molecules of Co2 (2Co2 at step-3 and 2Co2 at step-4 )
4) 2 ATP at step-5 by substrate level phosphorylation

41. Regulation of the Citric Acid Cycle
 The citric acid cycle must be carefully regulated by the cell.
 If the citric acid cycle were permitted to run unchecked, large
amounts of metabolic energy would be wasted in the over
production of reduced coenzymes and ATP.
 Conversely if the citric acid cycle ran too slowly, ATP would not
be generated fast enough to sustain the cell.
 By looking at the changes in free energy of the reactions of the
citric acid cycle, it is clear that there are three irreversible steps.
 the three enzymes of the reactions of the cycle: citrate synthase
(step-1), isocitrate dehydrogenase (step-3) and α-ketoglutarate
dehydrogenase (step-4) operate with large negative free energy
changes under the concentrations of products and reactants in the
matrix of the mitochondria and they are the regulated steps in the
TCA cycle

42.  The citric acid cycle is regulated by three simple mechanisms.
These are:
1. Substrate availability
2. Product inhibition
3. Competitive feedback inhibition.
 The principle signals for TCA cycle regulation are acetyl-CoA,
succinyl-CoA, ATP,ADP, AMP, NAD+ and NADH.
 All of the regulatory enzymes of the citric acid cycle including
pyruvate dehydrogenase are allosterically inhibited by NADH.
 The combination of the electron transport chain and oxidative
phosphorylation produce ATP form NADH, consequently ATP is
an allosteric inhibitor of pyruvate dehydrogenase and isocitrate
dehydrogenase.
 The TCA cycle is turned on however by high ratios of either
ADP/ATP or NAD+/ NADH which indicate that the cell has run
low of NADH or ATP.

43. Step-1(citrate
synthase
regulation)
Step-3
(isocitrate
dehydrogenase
regulation)
Step-4 (a-ketoglutarate
dehydrogenase regulation)
pyruvate
dehydrogenase
regulation

44. 4. The Electron Transport Chain(ETC)
 The citric acid cycle oxidizes acetate into two molecules of CO2 while
capturing the electrons in the form of NADH molecules and one
molecule of FADH2
 These reduced molecules contain a pair of electrons with a high
transfer potential.
 These electrons are ultimately going to be transferred by a system of
electron carriers to O2 to form H2O.
 This process occurs in the mitochondrial inner membrane and
 it is the major energy source used to produce ATP by oxidative
phosphorylation (the process of making ATP by using the proton
gradient generated by the ETC).
 About 32 or 34 ATP are produced in ETC by oxidative phosphorylation
 ETC is final stages of aerobic oxidation of biomolecules in
eukaryotes occur in the mitochondrion.
 The final electron acceptor in ETC is oxygen