Enzymes are usually proteins that act as catalysts and speed up biochemical reactions without being consumed. They have optimal temperatures and pH levels for activity. The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration. Competitive inhibitors bind the enzyme's active site, increasing Km; noncompetitive inhibitors bind elsewhere, decreasing Vmax. Enzymes are important clinically as markers of tissue damage - creatine kinase and lactate dehydrogenase indicate heart attacks, while alanine transaminase, aspartate transaminase and alkaline phosphatase detect liver disease. Troponin is a very specific marker for myocardial infarction.

1. Enzymology

2. • Biocatalyst
• Usually proteins except ribozyme (RNA particles with catalytic activity
)
• With out being changed themselves

3. Enzyme activity
Unit of enzyme activity • Amount causing transformation of 1 um of
substrate /min at 25 *C
• Expressed as mole of substrate utilised or mole of
product formed
Specific activity • No of protein units /mg of protein
• Measure of enzyme purity
• Higher the enzyme purity  higher the specific
activity
Turn over number • Number of substrate molecules transformed per
unit time by a single enzyme molecule (when
enzyme concentration alone is limiting factor)
• Highest turnover  catalase (fastest enzyme)
followed by carbonic anhydrase
• Lowest turnover  lysozyme (slowest enzyme)

4. Some enzymes are produced as proenzymes
• Inactive precursors  zymogens or proenzymes
• Proelastase
• Pepsinogen

6. Co enzyme
• organic molecule required by
enzyme
Co factor
• Inorganic molecule required by
enzyme

13. Nomenclature of enzyme
• EC stands for enzyme commission, and
• the first digit stands for the class name (transferase),
• the second digit stands for the subclass
• 3rd digit sub sub class
• 4th digit  individual enzyme

14. Isomerases
• Decarboxylases
• Aldolases

15. Ligases
• Synthetase
• Carboxylases
• Ligases

17. Co enzymes
• Water soluble vitamins
• Can function either as co substrate or prosthetic group

20. Sulfite oxidase

21. Molybdenum
• Xanthine oxidase
• Sulfite oxidase

22. Mechanism of enzyme action

23. • Most enzyme – substrate
combinations are mostly d/t weak
non covalent modification like
hydrogen bond hydrophobic
interactions & van der waal forces
• Increase the rate of biochemical
reaction
• Lowering the magnitude of the
activation energy barrier
• decreasining free energy of activation

24. Lowering of activation energy

25. Michaelis menten theory
Enzyme combines with a substrate to form a transient enzyme
substrate complex
Which break in to enzyme products

26. Temperature & enzyme activity
Rate of enzyme activity increases with increase in temperature d/t
increase in kinetic energy

27. Enzyme & pH

28. • Rate of reaction
increases directly with
increase in enzyme
cncentration

29. Hyperbolic curve is obtained

32. Michaelis menten equation
• Reaction velocity varies with substrate
concentration
• V0initial velocity
• Vmax  maximum velocity
• Km michaelis menten constant
• [S] substrate concentration

33. Km (michaelis menten constant)
• Substrate concentration at which reaction rate is half maximum
• Constant for each enzyme
• Reflects binding affinity of the enzyme for its substrate
• High enzyme substrate affinity implies a low Km value
• Low affinity implies high Km
• Natural substrate has lowest km
• key enzyme has highest km

34. Lineweaver burk plot

36. Lineweaver burk plot is also known as double
reciprocal plot

37. Fischers lock and key theory

38. Koshlands induced fit theory

40. Enzyme inhibition

41. Enzyme inhibition
• Competitive inhibition
• Noncompetitive inhibition
• Suicide inhibition
• Allosteric inhibition
• Feedback inhibition

42. Competitive inhibition
• Inhibitor is structural analog of substrate
• Binds to same site as substrate
• Km increases
• Reversible
• excess substrate abolishes inhibition
• Vmax remains same
• Km increase

44. Vmax remains same
Km increases (decreased affinity to substrate)

45. • Competitive inhibition
• Eg
• Statin on HMG coA reductase
• MTX on DHFR
• Dicumarol in vitamin K epoxide
• Succinate dehydrogenase by malonate

47. • Effect on Vmax:
• The effect of a competitive inhibitor is reversed by increasing [S]. At a
sufficiently high substrate concentration, the reaction velocity reaches the
Vmax observed in the absence of inhibitor .
• Effect on Km:
• A competitive inhibitor increases the apparent Km for a given substrate. This
means that, in the presence of a competitive inhibitor, more substrate is
needed to achieve 1⁄2Vmax.

48. Non competitive inhibition
• Can be reversible or irreversible
• Mostly irreversible
• Inhibitor have no structural similarity to
substrate
• Bind to site other than substrate binding site
• Affinity to substrate same  Km remains same
• Excess substrate donot abolish inhibition
• Vmax decreases
• Less enzyme activity

49. • 1. Effect on Vmax:
• Noncompetitive inhibition cannot be overcome by increasing the
concentration of substrate. Thus, noncompetitive inhibitors decrease the
apparent Vmax of the reaction.
• 2. Effect on Km:
• Noncompetitive inhibitors do not interfere with the binding of substrate to
enzyme. Thus, the enzyme shows the same Km in the presence or absence of
the noncompetitive inhibitor.

50. Non competitive inhibition
• Cyanide on cytochrome oxidase

53. Vmax remains same
Km increases (decreased affinity to substrate)

56. Uncompetitive inhibitor
• Bind only to enzyme substrate
complex  ESI complex
• Decrease in both Vmax
• Km reduced
• Phenylalanine & placental ALP

58. Suicide inhibition
• Mechanism based inactivation
• Inhibitors are unreactive until they are converted by enzyme in to
active product
• Which in turn inhibits the enzyme
• Eg aspirin COX
• Allopurinol xanthine oxidase

59. Suicide inhibition by aspirin

60. Feed back inhibition
• End product inhibition

61. Regulation of enzymes

62. • Regulation of enzyme quality
• Allosteric regulation
• Covalent modification
• Regualtion of enzyme quality
• Control of enzyme synthesis  induction & repression
• Control of enzyme degradation

63. Allosteric regulation
• Allosteric enzymes have a separate site where a modifier binds other
site for binding of substrates
• Can be
• Allosteric activator
• Allosteric inhibitor

66. • Allosteric enzymes have a quarternary structure & made up of sub
units

67. Cooperative binding  sigmoid shaped curve

68. Hills equation describes the allosteric
modification

69. Covalent modification

70. Covalent modification
• Reversible
• Phosphorylation /dephophorylation
• Methylation
• Adenylation
• Acetylation
• Irreversible
• Partial proteolysis /zymogen activation

71. Phosphorylation  most common covalent
modification
Enzymes active in phosphorylated
state
• Glycogen phosphorylase
• Key enzymes of gluconeogenesis
Enzymes active in dephosphorylated
sate
• Glycogen synthase
• Enzymes of glycolysis

72. Caspaces

73. • Catalase
• Highest turn over
• Fastest acting enzyme

74. • Serine Proteases
• They are enzymes with a serine residue at the active site and most of the
proteolytic enzymes belong to this group, e.g. trypsin, chymotrypsin, clotting
factors

75. Clinical enzymology

76. • Nonfunctional plasma enzymes
• Cell derived enzymes
• Released in to plasma by autolysis (necrosis) or increased permeability of cells

77. • Functional enzyme in plasma
• Clotting factors in plasma

78. Enzymes in Heart
• CK
• LDH
• AST

79. CK MB
• First enzyme to elevate
• Rises in 3- 8 hrs
• Remain elevated for 3 days
• Early diagnosis of MI

80. CK is a dimer made up of 2 subunits B & M
subunits

82. Fetal reversion
• Damaged skeletal muscle may contain more CK-MB owing to
phenomenon of fetal reversion, thus serum CK-MB isoenzyme may
increase in such conditions.
• In c/c muscle disorders

83. LDH
• Tetramer made of H & M subunits
• 5 isoenzymes
• Normally in blood LDH2 > LDH1
• But in MI  LDH 1 > LDH2 } FLIPPED PATTERN

84. • LDH
• Elevated after 12- 18 hrs (last enzyme to elevate)
• Peak value in 3 days
• Remain elevated for 14 days late diagnosis of MI

85. LDH

86. • LDH 2 is elevated in anemia

87. • LDH may also be elevated in haemolysis

88. AST
• Elevated in MI & hepatocellular damage
• Non specific
• Elevated after days & lasts for less duration
• Not used in diagnosis

90. Pro BNP
• The best marker of ventricular dysfunction is pro-BNP.

91. Ischemia modifed albumin (IMA)
• Myocardial ischemia alters the N-terminus of albumin reducing the
ability of cobalt to bind to albumin.
• Rises with in 6 – 10 minutes
• Low specificity (ischemia in any organ)
• negative value is highly useful, as it rules out the possibility of MI

92. Myocardial ischemia alters the N-terminus of
albumin reducing the ability of cobalt to bind to
albumin

93. Myoglobin
• Low specificity

94. • Troponin I
• is released into the blood within 4 hours after the onset of symptoms of
myocardial ischemia; peaks at 14–24 hours and remains elevated for 3–5 days
postinfarction
• Troponin T (TnT)
• increases within 6 hours of myocardial infarction, peaks at 72 hours and then
remains elevated up to 10–14 days

95. Troponin
• Skeletal muscle cardiac muscle
• Not in smooth muscle
• Any time marker for MI

98. H -FABP
• Heart type fatty acid binding protein
• Used as predictor of death or MI @ 1 yr in ACS
• H FABP – ve  low mortality
• H FABP +ve  high mortality

99. Enzymes in liver disease

100. Enzymes in liver disease
• Hepatocellular damge
• ALT
• AST
• Obstructive
• ALP
• Gamma glutamyl transferase
• 5 nucleotidase

101. AST / ALT
• AST /ALT <1 :viral hepatitis
• AST /ALT 1-2 : cirrhosis
• AST /ALT >2 : alcoholic hepatitis or HCC

102. Isoenzymes of ALP
• Alpha 1 ALP-
• epithelial cells of biliary canaliculi
• increased in obstructive jaundice
• Alpha 2 heat labile ALP-
• hepatic cells
• Moderate elevation in jaundice
• Alpha 2 heat stable ALP placental
• -not destroyed at 65˚C inhibited by phenylalanine
• Pre beta ALP – bone,heat labile
• Gamma ALP –
• intestinal cells inhibited by phenylalanine
• Elevated in ulcerative colitis
• Leukocyte alkaline phosphatase
• –decreased in CML increase in lymphoma

103. • ATYPICAL ISOENZYMES
• Regan isoenzyme-heat stable,inhibited by L-phenylalanine
• a/w ca liver lung GIT
• Also increased in smokers
• Nagao isoenzyme- variant of regan inhibited by L-leucine
• In pleural malignancy

104. 5 ‘ nucleotidase
• 2-17 U/L
• Elevated in obstructive ds

105. GGT (gamma glutamyl transferase )
• Very sensitive for alcoholic liver ds
• Present on membrane surface

106. Pancreatic ds
• S amylase
• Non specific
• Increased in all cases of a/c abdomen
• Small protein excreted in urine  elevated in renal failure
• S lipase
• Specific

107. Prostate
• Acid phosphatase
• PSA

108. Acid phosphatase
• Secreted by prostate cells, RBC, platelets and WBC.
• prostate iso-enzyme is inactivated by tartaric acid
• Erythrocytic form is inhibited by cupric ions
• increased in prostate cancer and highly elevated in bone metastasis of
prostate cancer
• tartrate labile iso-enzyme is elevated.
• This assay is very helpful in follow-up of treatment of prostate cancers.
Tartrate resistant acid phosphatase
• Elevated in osteoclastoma
• Osteodystrophy
• Metabolic bone ds

109. PROSTATE SPECIFIC ANTIGEN (PSA)
• serine protease
• Normal value is 1–5 mg/L.
• It is very specifc for prostate activity.
• Values above 10 mg/L is indicative of prostate cancer

110. Anti tranglutaminase activity
• In celiac ds
• Sensitve & specific

112. Abzyme
• Ab with enzymatic
activity