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
5.
6. Co enzyme
• organic molecule required by
enzyme
Co factor
• Inorganic molecule required by
enzyme
7.
8.
9.
10.
11.
12.
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
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
32. Michaelis menten equation
• Reaction velocity varies with substrate
concentration
• V0initial 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
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
45. • Competitive inhibition
• Eg
• Statin on HMG coA reductase
• MTX on DHFR
• Dicumarol in vitamin K epoxide
• Succinate dehydrogenase by malonate
46.
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.
56. Uncompetitive inhibitor
• Bind only to enzyme substrate
complex ESI complex
• Decrease in both Vmax
• Km reduced
• Phenylalanine & placental ALP
57.
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
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
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
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
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
81.
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
88. AST
• Elevated in MI & hepatocellular damage
• Non specific
• Elevated after days & lasts for less duration
• Not used in diagnosis
89.
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
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
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
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
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
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