2. Prosthetic groups, Cofactors and coenzymes
2
Some enzymes require no chemical groups for activity
Many enzymes contain small nonprotein molecules and metal ions that
participate directly in substrate binding or catalysis.
• Prosthetic groups
• Cofactors
• Coenzymes
3. Metal ions as Prosthetic groups or Cofactors
3
Certain mechanisms as to how the metal ions bring about activation:
Direct participation in catalysis
Formation of a metallosubstrate
Formation of a metalloenzyme
Alteration of equilibrium constant
Conformational change in the enzyme
4. Prosthetic groups
4
Prosthetic groups are tightly and stably incorporated into a protein’s structure.
Examples:
• Pyridoxal phosphate
• Flavin mononucleotide (FMN)
• Flavin adenine dinucleotide (FAD)
• Thiamin pyrophosphate
• Biotin
• Metal ions (Co, Cu, Mg, Mn, & Zn)
5. Prosthetic groups
The most common → Metals
~One-third of all enzymes → metalloenzymes
Metals:
Facilitate the binding and orientation of substrates.
Facilitate the formation of covalent bonds with reaction intermediates (co2+ in
coenzyme B12).
Interact with substrates to render them more electrophilic/nucleophilic.
6. Cofactors
6
Cofactors associate reversibly with enzymes or substrates.
Functions similar to those of prosthetic groups.
But bind in a transient,dissociable manner either to the enzyme/a substrate such as ATP.
Most are metal ions
Enzymes that require a metal ion cofactor →metal-activated enzymes
8. Coenzymes
8
Coenzymes serve as Recyclable shuttles:
Substrate shuttles
Group transfer agents:
• Methyl groups (folates)
• Acyl groups (coenzyme A)
• Oligosaccharides
10. • Enzyme concentration
• Substrate concentration
• Temperature
• pH
• Presence of inhibitors/ activators/cofactors
• Electrolyte concentrations
• Specific kinetic properties of the enzyme
• Other factors: intracellular compartmentation/ local microenvironment
Factors affecting enzyme activity
10
11. 1) Substrate concentration:
Rate of enzyme catalyzed reaction increases
with increase in [S] until the maximal
velocity (Vmax) is reached.
NB: afterVmax is reached increasing the [S]
will not have any effect on reaction rate due
to saturation (active sites are occupied by
reactants forming ES).
Factors Affecting rate of the reaction
Effect of substrate concentration on the initial
velocity of an enzyme-catalyzed reaction
11
12. 2)Temperature:
Increase inT increases reaction rate to reach maximum activity due to increased
number of molecules having sufficient energy.
but further increase lowers the rate due to denaturation of the enzyme (loss of
native conformation hence loss of activity)
TheT at which the enzyme is 100% active is termed the optimum
temperature.
Factors affecting….
12
13. 3) pH
The same toT, PH is required for enzyme activity.
but further increase in PH due to denaturation of enzyme brings in sharp decline in
enzyme activity hence reaction rate also decreases.
NB:The pH at which an enzyme is 100% active is termed the optimum PH.
The optimum PH varies for different enzymes.
Some are active at neutral/acidic & Others at neutral PH.
4) Inhibitors also affect enzyme activity.
Factors affecting …
13
14. pH optima for different enzymes (left),and temperature optima for DNA polymerase 1
(Pol1) andTaq (Thermophilus aquaticus) DNA polymerase (right)
14
15. Enzymes (E) + Substrates (S) ⇌ E-S complex ⇌E +P
Enzyme-substrate interactions are generally non-covalent
But chemical bonds are broken and made during an enzyme-catalysed reaction.
Enzyme kinetics
16. 16
Enzyme kinetics………………con’t
The transition state is an unstable “intermediate” during a reaction leading to product
formation.
– Energy is needed to form the transition state.
– Enzymes lower the energy needed to form the transition state.
–comes from binding of substrate to the enzyme
17. Enzyme kinetics………………con’t
Enzymes do not change the overall free energy change of a reaction.
− Do change the energy of activation.
Transition state
17
18. They postulated that the enzyme first combines reversibly with its substrate to form an E-S
complex in a relatively fast reversible step:
The ES complex then breaks down in a slower 2nd step to yield the free E & the reaction
product P.
Because the slower second reaction must limit the rate of the overall reaction, the overall
rate must be proportional to the concentration of the species that reacts in the second step,
that is, ES.
E = enzyme, S = substrate, ES = enzyme-substrate complex, P = product
K1, k-1, k2 & k-2 are rate constants & Km= (k-1+ k2)/k1, where Km =Michaelis
constant
Leonor Michaelis and Maud Menten in 1913
18
19. The Michaelis-Menten Equation:
19
Where,
Vo is the initial velocity
Vmax is the maximal velocity
[S] is the substrate concentration
Km is called the Michaelis Constant
Km is the [S] at (Vmax/2).
related to the affinity of an enzyme for
its substrate.
Michaelis-Menten Enzyme Kinetics
22. Types of enzyme inhibitors
22
Reversible and irreversible inhibitors:
•Most,but not all,drugs are reversible inhibitors.
•Generally speaking,reversible inhibitors bind non-covalently to enzyme.
• Examples of irreversible inhibitors:
•Aspirin (cox 1 and 2 inhibitor)
•Penicillins (inhibit dd-transpeptidase and disrupt peptidoglycan of bacterial cell walls)
•Organophosphate (acetylcholinesterase inhibitors)
•Examples of reversible inhibitors:
•Statins (LDL cholesterol-lowering drugs)
•Peptide-based HIV protease inhibitors
•Influenza virus neuraminidase inhibitors.
23. 23
Irreversible inhibitors of enzymes:
ASPIRIN (acetylsalicylic acid): an irreversible inhibitor of cyclooxygenase enzymes.
Prostaglandin and thromboxane biosynthesis
Involved in numerous processes, including inflammation, body temperature control, pain
sensation, blood clotting.
Aspirin is an anti-inflammatory, analgesic and antipyretic drug and reduces the clotting ability of blood.
(Pg= prostaglandin, tx= thromboxane).
ASPIRIN
24. Aspirin covalently reacts with a serine residue side-chain of cyclooxygenase,
inactivating the enzyme irreversibly.
E= enzyme [cyclo-oxygenase]
SER-OH = serine residue in the enzyme’s active site
CH3
24
25. Irreversible enzyme inhibitors: Penicillins ( and other beta-lactams).
The beta-lactams resemble the chemical structure of D-Ala-D-Ala found in peptides in the
peptidoglycan of the bacterial cell wall.
25
26. 26
Penicillins:
Transpeptidases (penicillin-binding protein, PBP) cross-link the peptidoglycan bacterial
cell wall structure.
cross-linking between D-Ala of one peptide chain to the DAP (diaminopimelic acid) of
another chain.
The enzyme is Irreversibly inactivated by Penicillin and the bacteria rupture.
28. Irreversible enzyme inhibitors: Organophosphate acetylcholinesterase inhibitors:
28
Some insecticides (e.g. malathion) and nerve toxins (e.g. sarin) are organophosphates.
They irreversibly inactivate acetylcholinesterase.
• Prevent the breakdown of the neurotransmitter, acetylcholine.
• Causing many neurotoxic symptoms due to excessive acetylcholine levels.
Symptoms include:
• Paralysis
• Muscle cramps
• Weakness
• Elevated blood pressure
• Seizures
• Ataxia
• Respiratory muscle failure
• Salivation
• Lacrimation
• Sweating
Death is usually due to respiratory muscle paralysis and can occur within minutes of
exposure.
29. *There are three main types of reversible inhibition:
I. Competitive inhibitors
II. Uncompetitive inhibitors
III. Noncompetitive inhibitors
* They interact reversibly with enzyme or (ES)-complex at d/t stages of the reaction process.
29
30. I. Competitive inhibition:
The inhibitor binds to the active site of the enzyme and competes with the substrate for the active site.
The effect of the inhibitor can be overcome by excess substrate, soVmax is not affected. What about Km?
30
31. Example of reversible competitive enzyme inhibitor:
Statins competitively inhibit the rate-limiting enzyme of cholesterol biosynthesis:
HMG-CoA reductase (hydroxymethylglutaryl-CoenzymeA reductase).
They lower low density lipoprotein levels (LDL or “bad cholesterol” in blood in particular.
31
32. FightingAIDS with Inhibitors of HIV ReverseTranscriptase
32
HIV’s reverse transcriptase has a higher affinity for AZT triphosphate than for dTTP, and
binding ofAZT triphosphate to this enzyme competitively inhibits dTTP binding.
WhenAZT is added to the 3’ end of the growing DNA strand, lack of a 3’ hydroxyl means
that the DNA strand is terminated prematurely and viral DNA synthesis stops.
34. Reversible competitive inhibitors:
Influenza neuraminidase inhibitors inhibit competitively the neuraminidase of the virus.
This enzyme is present on the surface of the virus for its entry into human cells.
Neuraminidase inhibitors (e.g. oseltamivir) are used to treat acute influenza virus infections.
34
35. II. Uncompetitive inhibition:The inhibitor can bind only when the substrate is already
bound to the enzyme. Both Km andVmax are decreased.
35
36. III. Noncompetitive Inhibition:
The inhibitor binds to an E to form an EI- complex, and/or to an ES- complex to form an ESI.
The inhibitor binds away from the active site, and the affinity of enzyme for substrate is not affected.
The reaction is slowed down by the inhibitor, and this cannot be overcome by excess substrate.
Vmax is decreased and Km is unchanged.
Noncompetitive inhibition
36
41. 41
Constitutive enzymes are required at all times by a cell and present at some constant
level.
• Housekeeping enzymes
• For example, many enzymes of the central metabolic pathways.
Regulatory enzymes:
Increased or decreased catalytic activity.
42. Regulatory Enzymes
42
Allosteric enzymes are regulated by:
Allosteric effectors/modulator.
Allosteric effector-mediated regulation
Generally small metabolites or cofactors
Other enzymes are regulated by reversible covalent modification.
Some enzymes are stimulated/inhibited when they are bound by separate regulatory
proteins.
Others enzymes are also activated when peptide segments are removed by proteolytic
cleavage
⇛ irreversible
⇛E.g., Zymogens
43. Regulatory Enzymes:Allosteric enzymes
43
Enzymes with several modulators generally have different specific binding sites for each.
The modulators for allosteric enzymes may be inhibitory/stimulatory.
Often the modulator is the substrate itself.
The regulation in which substrate and modulator are identical ⇨homotropic enzymes.
o Active site and regulatory site are the same.
When the modulator is a molecule other than the substrate ⇨ heterotropic enzyme
• E.g., PFK1 ⇦ADP,AMP,ATP & Citrate
44. Regulatory Enzymes:Allosteric enzymes
44
Feedback regulation:
Feedback inhibition: Inhibition of an allosteric enzyme
at the beginning of a metabolic sequence by the end
product of the sequence.
Also known as end-product inhibition
Eg. Regulation of pyruvate dehydrogenase (PDH)
45. Regulatory Enzymes
45
Feedback regulation is not synonymous with feedback inhibition.
For example, dietary cholesterol decreases hepatic synthesis of
cholesterol.
This feedback regulation does not involve feedback inhibition.
Regulation of cholesterol formation balances
synthesis with dietary uptake and energy state
46. Regulatory Enzymes
46
Enzymes modulated by covalent modification
Over 500 different types of covalent modification
Reversible
one or more of its amino acid residues
Like allosteric enzymes, they tend to be multisubunit proteins.
In some cases the regulatory site(s) and the active site are on separate subunits.
49. 49
Examples of mammalian enzymes whose catalytic activity is altered by
covalent phosphorylation-dephosphorylation.
50. Enzymes regulated by proteolytic cleavage
50
Some enzymes are regulated by proteolytic cleavage of an enzyme precursor.
An inactive precursor zymogen is cleaved to form the active enzyme.
Specific cleavage causes conformational changes that expose the enzyme active site.
o E.g., proteases of the stomach and pancrease
o Chymotrypsin & trypsin are initially synthesized as chymotrypsinogen & trypsinogen
51. Activation of zymogens by proteolytic cleavage
51
The three polypeptide chains (A, B, and C) of chymotrypsin
are linked by disulfide bonds
52. Control of Enzyme Synthesis
52
Inducers stimulate the transcription of the gene that encodes regulatory enzymes.
Or transcription factors
Conversely, an excess of a metabolite may act as repressors for repression of
synthesis of regulatory enzymes.
53. Clinical Enzymology in Diagnosis of Disease
Specific enzymes for diagnosis and prognosis of disease.
Deficiencies in the quantity or catalytic activity.
Genetic defects (genetic mutations/infection by viral/bacterial pathogens)
Nutritional deficits
Toxins
Assaying the activity of specific enzymes:
• Blood
• Other tissue fluids
• Cell extracts
53
54. Enzymes as diagnostic biomarkers
1) Functional plasma enzymes (Plasma derived enzymes)
Certain enzymes, proenzymes, and their substrates are present at all times in the
circulation
perform a physiologic function in the blood.
Examples:
• Lipoprotein lipase
• The proenzymes of blood coagulation
The majority are synthesized in and secreted by the liver.
54
55. Enzymes as diagnostic biomarkers
2) Nonfunctional plasma enzymes (Cell derived enzymes)
Plasma also contains numerous other enzymes that perform no known physiologic function in
blood.
Arise from the routine normal destruction of erythrocytes,leukocytes,and other cells.
Tissue damage or necrosis resulting from injury or disease increases their plasma levels
55
56. Enzymes as diagnostic biomarkers
In damaged, infected or inflamed tissue:
Cell membranes become more permeable/destroyed.
The content of the cell cytoplasm → extracellular space → bloodstream
The enzymatic activity can be measured.
Useful in the diagnosis of damage to the heart, liver, muscle, etc.
56
57. Enzymes as diagnostic biomarkers
Unit of serum enzyme activities:
International unit (IU) :
One IU is defined as the activity of the enzyme which transforms one micro mole of
substrate into products per minute per liter of sample under optimal conditions and at
defined temperature .
It is expressed as IU/L
Katal – catalytic unit
One Katal is defined as the number of mole of substrate converted to product
per second per liter of sample.
57
58. Enzymes as diagnostic biomarkers
Enzyme estimations are helpful in the diagnosis of –
Myocardial Infarction
Liver diseases
Muscle diseases
Bone diseases
Cancers
GITract diseases
58
59. Principal Serum Enzymes Used in Clinical Diagnosis
Note:Many of the above enzymes are not specific to the disease list
59
60. Enzyme estimations for diagnosis of Acute Myocaridal Infraction (AMI)
Enzyme assays routinely carried out for the diagnosis of AMI includes;
• Creatine kinase
• Aminotransferases → AST (SGOT) and ALT (SGPT)
• Lactate dehydrogenase
• Cardiac troponin (protein)
60
61. Enzyme estimations for diagnosis of AMI
After a heart attack,a variety of enzymes leak from injured heart cells into the bloodstream.
Blood serum concentrations ALT,AST and CK can be measured by:
• SGPT, SGOT and SCK tests, respectively.
Lactate dehydrogenase also leaks from injured or anaerobic heart muscle.
Provide information about the severity of the damage.
61
62. Creatine kinase
Found in all the muscles of the body.
But much more in skeletal muscle than cardiac muscle.
However, CK activity can be due to one of three isoenzymes
CK1 or CKMM, found mostly in skeletal muscle
CK2 or CKMB, found predominately in cardiac muscle
CK3 or CKBB, found in smooth muscle
Further isoforms of CKMM and CKMB:
Two isoforms of CKMB →MB1 and MB2
Three isoforms of CKMM → MM1, MM2, and MM3
62
63. Lactate dehydrogenase (LDH)
It has at least five different isozymes.
All isozymes contain 4 polypeptide chains.
The M (for muscle) chain and the H (for heart) chain are encoded by two different genes.
63
64. Lipase
Secreted by pancreas and Liver
Hydrolysis of fats
The plasma lipase level may be low in liver disease,Vitamin A deficiency, some
malignancies, and diabetes mellitus.
It may be elevated in acute pancreatitis and pancreatic carcinoma.
64
65. α- Amylase
Dietary starch and glycogen to maltose
Pancreatic juice, saliva, liver fallopian tubes and muscles
Mainly used in the diagnosis of acute pancreatitis.
The plasma amylase level may be low in liver disease
• But increased in high intestinal obstruction,mumps,acute pancreatitis and diabetes.
Trypsin
Secreted by pancreas
Elevated levels in plasma occur during acute pancreatic disease
65
66. Test Abnormality
Serum aminotransferases, for exampleAST,ALT Parenchymal injury
Serum bilirubin Cholestasis
Alkaline phosphatase, γ-glutamyl transferase Biliary epithelial damage and biliary
obstruction (alcohol)
Serum albumin Synthetic function
Prothrombin time (INR) Clotting (dynamic indicator)
Common liver function tests and the abnormalities they detect.
Liver function tests
66
Routine liver function tests (widely performed groups of serum measurements)
67. Liver function tests → Aminotransferases
Aminotransferases (transaminases)
Aspartate aminotransferase (AST) and alanine aminotransferase (ALT)
Their activities are typically 3,000–7,000-fold higher in hepatocytes than in plasma.
Commonly used as a test of hepatocellular damage.
AST is found in liver, heart, skeletal muscle, kidney, brain, and red blood cells.
ALT has a similar distribution, its concentrations are lower in extrahepatic tissues.
67
68. Liver function tests → Aminotransferases
AST exists in both cytosol and mitochondria.
But ALT is in a cytosolic form only.
Reference ranges for AST and ALT are 5–45 IU/L
Increased AST/ALT activity due to liver disease.
An increase in [transaminases] with a relatively smaller increase in other tests indicates
hepatitic liver disease:
I. Infective agents
II. Toxins
III. Autoimmune disorders
68
69. Liver function tests → Aminotransferases
I. Infective agents:
• Hepatitis A/B/C
• Cytomegalovirus (CMV)
• Epstein-barr virus (EBV)
• Others both viral and bacterial
• All produce a significant increase in serum AST/ALT.
ALT is better thanAST for monitoring viral activity in chronic hepatitis B or C.
The higher the relative increase in AST compared to ALP →hepatitis
69
70. Liver function tests → Aminotransferases
II.Toxins
↑AST/ ALT
Alcoholic liver damage is usually chronic and cholestatic (cirrhotic)
• AST/ALT ratio greater than two is suggestive of alcohol misuse.
Many drugs ↑AST/ ALT due to hepatocyte destruction.
• overdosage of paracetamol
Metals can be toxic to the liver & ↑AST/ ALT in the serum.
• An inherited metabolic diseases Cu (Wilson’s disease) & Fe (haemochromatosis)
There is a direct relationship b/n the serum activities of AST and ALT and BMI.
BMI above 30 can increase both by 40–50%.
70
71. Other secondary biochemical liver tests
• Albumin
• Globulins
• Alpha-fetoprotein (AFP)
• Carbohydrate deficient transferrin
• The carbohydrate antigen CA19-9
• Copper/caeruloplasmin
•α1-antitrypsin
71
