Enzymes catalyze biochemical reactions and are characterized by their catalytic activity. The document discusses several key aspects of enzyme mechanisms and kinetics, including:
1) Enzymes have an active site that binds substrates and facilitates chemical reactions through proximity, orientation, and catalytic groups like metal ions or amino acid residues.
2) Michaelis-Menten kinetics describe the relationship between substrate concentration, reaction rate, and parameters like Vmax and Km.
3) Common metalloenzymes contain catalytic metal ions like zinc, copper, iron and manganese that participate in redox reactions.
The flux of metabolites through metabolic pathways involves
catalysis by numerous enzymes. Active control of homeostasis is achieved by the regulation of only a small number of enzymes.
Active sites of the enzyme is that point where substrate molecule bind for the chemical reaction. It is generally found on the surface of enzyme and in some enzyme it is a “Pit” like structure
The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence
The active site takes up a relatively small part of the total volume of an enzyme
Active sites are clefts or crevices
Substrates are bound to enzymes by multiple weak attractions.
The specificity of binding depends on the precisely defined arrangement of atoms in an active site.
The flux of metabolites through metabolic pathways involves
catalysis by numerous enzymes. Active control of homeostasis is achieved by the regulation of only a small number of enzymes.
Active sites of the enzyme is that point where substrate molecule bind for the chemical reaction. It is generally found on the surface of enzyme and in some enzyme it is a “Pit” like structure
The active site is a three-dimensional cleft formed by groups that come from different parts of the amino acid sequence
The active site takes up a relatively small part of the total volume of an enzyme
Active sites are clefts or crevices
Substrates are bound to enzymes by multiple weak attractions.
The specificity of binding depends on the precisely defined arrangement of atoms in an active site.
This ppt describes the overview of enzyme regulation and Allosterism. Presented since October 23,2017GC at Addis Ababa University, School of Medicine, Department of medical biochemistry.
The rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into the product(s) in a specified time period.
Numerous factors affect the rate of enzyme-catalyzed reactions.
activation energy of biological systemKAUSHAL SAHU
SOME GENERAL TERM
FREE ENERGY
ENDERGONIC REACTION
EXERGONIC REACTION
ACTIVATION ENERGY
DEFINITION
TRANSITION STATE
WHERE DOES ACTIVATION ENERGY COME FROM?
DETERMINING THE ACTIVATION ENERGY THROUGH ARREHINIUS EQUATION
EFFECTS OF TEMPERATURE ON ACTIVATION ENERGY
NEGATIVE ACTIVATION ENERGY
EFFECTS OF ENZYMES ON ACTIVATION ENERGY
CONCLUSION
REFERENCES
Large family of proteolytic enzymes
All have serine residue at their active site which plays a crucial part in the enzymatic activity.
All cleave peptide bonds, by a similar mechanism of action. They differ in their specificity and regulation.
Serine proteases include:
the pancreatic proteases: trypsin, chymotrypsin and elastase,
various tissue/intracellular proteases such as leukocyte elastase
enzymes of the blood clotting cascade
some enzymes of complement system
Many serine proteases are synthesized as inactive precursors (zymogens) which are activated by proteolysis
Enzyme inhibition is explained with its kinetics, animations showing mechanism of inhibitors action, examples of inhibitors are explained in detail with Enzyme inhibited.
by Dr. N. Sivaranjani, MD
OBJECTIVE
The objective of this assignment is
• To have a detailed overview of Allosteric Enzymes
• To understand the mechanism and kinetics of Allosteric Enzyme
• To understand the importance of allosteric enzyme
This ppt describes the overview of enzyme regulation and Allosterism. Presented since October 23,2017GC at Addis Ababa University, School of Medicine, Department of medical biochemistry.
The rate of a chemical reaction is described by the number of molecules of reactant(s) that are converted into the product(s) in a specified time period.
Numerous factors affect the rate of enzyme-catalyzed reactions.
activation energy of biological systemKAUSHAL SAHU
SOME GENERAL TERM
FREE ENERGY
ENDERGONIC REACTION
EXERGONIC REACTION
ACTIVATION ENERGY
DEFINITION
TRANSITION STATE
WHERE DOES ACTIVATION ENERGY COME FROM?
DETERMINING THE ACTIVATION ENERGY THROUGH ARREHINIUS EQUATION
EFFECTS OF TEMPERATURE ON ACTIVATION ENERGY
NEGATIVE ACTIVATION ENERGY
EFFECTS OF ENZYMES ON ACTIVATION ENERGY
CONCLUSION
REFERENCES
Large family of proteolytic enzymes
All have serine residue at their active site which plays a crucial part in the enzymatic activity.
All cleave peptide bonds, by a similar mechanism of action. They differ in their specificity and regulation.
Serine proteases include:
the pancreatic proteases: trypsin, chymotrypsin and elastase,
various tissue/intracellular proteases such as leukocyte elastase
enzymes of the blood clotting cascade
some enzymes of complement system
Many serine proteases are synthesized as inactive precursors (zymogens) which are activated by proteolysis
Enzyme inhibition is explained with its kinetics, animations showing mechanism of inhibitors action, examples of inhibitors are explained in detail with Enzyme inhibited.
by Dr. N. Sivaranjani, MD
OBJECTIVE
The objective of this assignment is
• To have a detailed overview of Allosteric Enzymes
• To understand the mechanism and kinetics of Allosteric Enzyme
• To understand the importance of allosteric enzyme
Drug screening assays for phosphate-generating enzymesExpedeon
Find out more about phosphate detection, ATPase and GTPase assays here: http://www.innovabiosciences.com/phosphate-detection.html
This presentation introduces drug screening assays with a particular focus on phosphate-generating enzymes such at ATPases/GTPases. Topics covered during the twenty minute presentation include:
1. Methods of phosphate detection
2. Application to assays for phosphate generating enzymes
3. Enzyme activity calculations
4. Malachite green assay
5. Improved malachite assays with greater stability/linear range
6. PiColorLock reagent -- advantages for High Throughput Screening
7. ATPase/GTPase assays
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This ppt includes overall idea of what is enzymes, how it works, mechanism of enzymes, kinetics and how to inhibit enzyme activities. The reference is the ideal book for biochemistry - Lehninger . Understanding is easy for everyone.
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1. Enzymes II Department of Bioche mistry (J.D.) 2011 Mechanism of enzym e rea ction , metal l oenzym es , kineti cs , a c tivit y , enzym es in medicine
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4. Example: The active sites of three proteinases Certain AA residues determin e the substrate specificity of these enzymes. The peptide bonds prefer ably hydrolyzed: by chymotrypsin - near residues with large, hydrophobic, uncharged side chains (Phe, Trp), by trypsin – near residues with long, positively charged side chains (Arg, Lys), by elastase - near residues with small hydrophobic side chains (Gly, Ala). A deep, relatively hydrophobic pocket At the bottom of the deep pocket there is an acidic residue (Asp) Two residues of valine close off the mouth of the small hydrophobic pocket
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6. Sequential reaction : - type ordered - the substrates bind the enzyme in a defined sequence - type random – the order of addition of substrates and release of products is random: Ping-pong reaction: + + +
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9. Example: active site of chymotrypsin Nucleophilic attack of serine -OH to carbonyl carbon of peptide bond serine protease 195 57 simplified
10. Active site of chymotrypsin: the cleavage of peptide bond
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21. Kinetic equation for rea ction S P v = k [S] = k [S] 1 reaction of 1. order k = rate c onstant
22. K inetic (progress) curve s for substrate and product during rea ction : concentra tion of substr ate decreases concentration of product increases reaction rate is determined from kinetic curves The instantaneous velocity v x at any particular time t x is g iven by the slope of the tangent to the curve at that time. [ P ] t [ S ] t (equilibrium)
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26. The graph of previous equation is saturation curve enzyme saturated by substrate
27. If [S] << K m at low substrate concentration the reaction proceeds by the 1 st order kinetics ×
28. If [S] >> K m at high substrate concentration the reaction proceeds by the 0. order kinetics ×
29. Two parts of saturation curve compare with pages 21 and 23 [ S ] V 0 V max K m The zero-order kinetics The 1 st order kinetics
32. From the obtained progress curves, the values v o are estimated and plotted against the corresponding [S] . T he velocity v o rises linearly as substrate concentration increases, and then begins to level till it reaches a limit value at high substrate concentrations. How to get a saturation curve? E = const . A series of measurements of initial velocity is arranged at a constant enzyme concentration E and different substrate concentrations S (in the range of 2 - 3 orders). [ P ] t [ S 1 ] [ S 2 ] [ S 3 ] [ S 4 ] v 0 1 v 0 2 v 0 3 v 0 4
36. Reciprocal form is the equation of a line ( y = a x + b) 1/ v o ................ dependent variable ( y ) 1/ [S] ............... independent variable ( x ) 1/ K m ........... .... 1/ V max ............. easily determined from the graph
38. Initial velocity depends on enzym e concentration [E] v o [S] [E 1 ] [E 2 ] > [E 1 ] [E 3 ] > [E 2 ] K M does not change, V max increases with increased [E] saturated enzym e : v o = k [E] t [E] t is tot al c oncentra tion of enzym e
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42. Distinguish: unit and its dimension mol/l.s kat/l Catalytic concentration mol/s kat Catalytic activity Dimension Unit Quantity
43. Determination of catalytic activity of ALT: two coupled enzyme reactions alanine + 2-oxoglutarate pyruvate + glutamate blood serum sample (ALT) pyridoxal-P pyruvate + NAHD+H + lactate + NAD + Δ A/ Δ t optical (UV) test Semin ars , p .18 the decrease of absorbance at 340 nm is followed in time LD
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45. Two methods for catalytic concentration less exact exact Evaluation of method average velocity initial velocity v o What is determined no yes Kinetic curve needed after some time (e.g. 10 min) the reaction is stopped by enzyme inactivation continually ( e.g. in 10 s econds ) How [P] [S] or [P] What is measured Constant-time method Kinetic method Feature
46. Problem 1 Enzyme sample (0.1 ml) was added to substrate solution. After 5 min, 0 . 2 mmol of produ ct was determined. What is catalytic concentration of enzyme?
47. Solution t = 5 min = 5 × 60 s = 300 s in 300 s … 0.2 mmol of product in 1 s … x = 0.2/300 = 6.7 × 10 -4 mmol / 0.1 ml of sample for 1 litre of sample = 6.7 × 10 -4 × 10 4 = 6.7 mmol/l.s = 6.7 mkat/l
48. Problem 2 Reaction mixture contains: 2.5 ml buffer 0.2 ml solution of NADH (optical UV test) 0.1 ml blood serum 0.2 ml substrate solution After 60 s, the decrease of NADH absorbance is A = 0.03 NADH = 6220 l/mol.cm, cuvette width l = 1 cm. What is catalytic concentration of enzyme?
49. Solution Serum sample was diluted: V final /V initial = 3,0 / 0,1 = 30 Lambert-Beer law: A = c l changes of absorbance and concentration expressed per time t A / t = c l / t t = 60 s Multiplied by dilution: 30 × 8 × 10 -8 = 2,4 × 10 -6 mol/l.s = 2,4 × 10 -6 kat/l = 2,4 kat/l
50. Consider that catalytic concentration = rate of chemical reaction kat /l = mol/l.s
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52. Example of i rreversible inhibition Diisopropyl fluorophosphate (and similar pesticides and nerve gases) inhibits acetylcholine esterase by phosphorylation of a crucial serine residue.
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54. Malonate competitively inhibit succinate dehydrogenase Examples: Methotrexate competitively inhibits active sites for tetrahydrofolate of the dihydrofolate reductase in the synthesis of purine and pyrimidine bases of nucleic acids. It is used to treat cancer. Methotrexate Tetrahydrofolate Malonate Succinate COO – CH 2 CH 2 COO – COO – CH 2 COO –
55. Methanol poisoning is treated by ethanol ethanol and methanol are similar molecules they compete for active site in enzyme alcohol dehydrogenase
56. Competitive inhibitors increase K m without any change in V max The V max can be reached even in the presence of inhibitor, but at much higher concentrations of [S] that have to overcome the competing inhibitor concentration. K m [S] v 0 V max K m 1 / v 0 1 / [S] 1 / V max - 1 / K m No inhibitor Competitive inhibitor
57. Non - competitive inhibition Non - competitive inhibitors bind to both free enzyme and enzyme-substrate complex, but in contrast to competitive inhibitors, not in the active site (the structure of inhibitor is distinct from th at of substrate ). Non - competitive inhibition cannot be overcome by increasing the substrate concentration. The non - inhibited remaining molecules of the enzyme behave like a more diluted solution of the enzyme.
58. Non - competitive inhibitors decrease V max without any change in K m 1/v o 1 / [S] 1 / V max - 1 / K m Non - competitive inhibitor No inhibitor K m [S] v 0 V max V max Non - competitive inhibitor
66. The consequences of phosphorylation/dephosphorylation in two antagonistic enzymes activated inhibited By dephosphorylation, enzyme is inhibited activated By phosphorylation, enzyme is glycogen synthesis from energy-rich UDP-glucose glycogen degradation by energy-poor phosphate (P i ) Enzyme catalyzes Glycogen synthase Glycogen phosphorylase Feature
69. Satura tion curve of allosteric enzym es is sigmoid al bez efektoru activation without effector inhibition
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72. Examples of enzymes in clinical diagnostics ALT alanine aminotransferase, CK creatine kinase, PSA prostate specific antigen In cell damages, activity of intracellular enzymes in extracellular fluid (blood serum) is elevated hepatopaties myopaties, myocardial infarction prostate cancer Elevation in serum indicates up to 0,9 kat/l up to 4 kat/l up to 4 μ g/l ALT CK PSA Reference values Enzyme
75. Enzymatic determination of glucose glucose + O 2 gluconolactone + H 2 O 2 H 2 O 2 + H 2 A 2 H 2 O + A glucose oxidase peroxidase colourless chromogen coloured product (absorbance measured) The principle of glucose determination in biochemical analyzers and in personal glucometers