Enzymes Introduction Kinetic reactions all chemical reactions in

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Enzymes

Enzymes

Introduction �Kinetic reactions: • all chemical reactions in living organisms require enzymes to work

Introduction �Kinetic reactions: • all chemical reactions in living organisms require enzymes to work For building , breaking down molecules or speed up the reactions. �Enzymes are : a biological catalyst. Usually a globular protein molecule produced by living organisms that can speed up a specific chemical reaction without itself being destroyed or changed in any way. �They are produced by the living organism and are usually present in only very small amounts in various cells.

Enzymes properties �Enzymes aren’t used up �Enzymes are not changed by the reaction •

Enzymes properties �Enzymes aren’t used up �Enzymes are not changed by the reaction • re-used again for the same reaction with other molecules. �Most enzymes are Proteins (tertiary and quaternary structures) �Act as Catalyst to accelerates a reaction � Are specific for what they will catalyze

Enzyme Kinetics Equation

Enzyme Kinetics Equation

The Lock and Key Hypothesis S E Enzyme-substrate complex Enzyme may be used again

The Lock and Key Hypothesis S E Enzyme-substrate complex Enzyme may be used again P P © 2007 Paul Billiet ODWS Reaction coordinate

Making réactions go faster o o Increasing the temperature make molecules move faster Biological

Making réactions go faster o o Increasing the temperature make molecules move faster Biological systems are very sensitive to temperature changes. Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy.

o o Enzymes can increase the rate of reactions without increasing the temperature. They

o o Enzymes can increase the rate of reactions without increasing the temperature. They do this by lowering the activation energy.

Factors affecting Enzymes o o substrate concentration p. H temperature inhibitors

Factors affecting Enzymes o o substrate concentration p. H temperature inhibitors

Substrate concentration: Reaction velocity Substrate concentration �The increase in velocity is proportional to the

Substrate concentration: Reaction velocity Substrate concentration �The increase in velocity is proportional to the substrate concentration © 2007 Paul Billiet ODWS

Substrate concentration: Vmax Reaction velocity Substrate concentration � Faster reaction but it reaches a

Substrate concentration: Vmax Reaction velocity Substrate concentration � Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied. Vmax © 2007 Paul Billiet ODWS

PH �Extreme p. H levels will produce denaturation �most human enzymes = p. H

PH �Extreme p. H levels will produce denaturation �most human enzymes = p. H 6 -8 �depends on where in body �pepsin (stomach) = p. H 3 �trypsin (small intestines) = p. H 8 �The structure of the enzyme is changed �The active site is distorted and the substrate molecules will no longer fit in it

Enzyme activity Trypsin Pepsin 1 3 5 7 p. H 9 11

Enzyme activity Trypsin Pepsin 1 3 5 7 p. H 9 11

Temperature �Effect on rates of enzyme activity �Optimum temperature �human enzymes � 35°- 40°C

Temperature �Effect on rates of enzyme activity �Optimum temperature �human enzymes � 35°- 40°C (body temp = 37°C) �Raise temperature (boiling) �denature protein = unfold = lose shape �Lower temperature T° �molecules move slower �fewer collisions between enzyme & substrate

Temperture and Enzymes ACTIVITY in human

Temperture and Enzymes ACTIVITY in human

�Michaelis and menten at low substrate concentrations, the enzyme is not saturated with the

�Michaelis and menten at low substrate concentrations, the enzyme is not saturated with the substrate and the reaction is not proceeding at maximum velocity whereas when the enzyme is saturated with substrate, maximum velocity is observed.

Inhibitors are chemicals that reduce the rate of enzymatic reactions. o They are usually

Inhibitors are chemicals that reduce the rate of enzymatic reactions. o They are usually specific and they work at low concentrations. o They block the enzyme but they do not usually destroy it. �Irreversible inhibitors: �Combine with the functional groups of the amino acids in the active site, irreversibly. o

Reversible inhibitors: There are two categories o 1. • • • Competitive: These compete

Reversible inhibitors: There are two categories o 1. • • • Competitive: These compete with the substrate molecules for the active site. The inhibitor’s action is proportional to its concentration. Come over these problem by adding more substrate

�Km : The addition of a competitive inhibitor increases the observed Km for a

�Km : The addition of a competitive inhibitor increases the observed Km for a given substrate. � Therefore, in the presence of a competitive inhibitor, more substrate is needed to achieve � Vmax: Competitive inhibitors do not alter Vmax. � The effect of a competitive inhibitor is reversed by increasing [S]. � high substrate concentration, the reaction velocity reaches the same Vmax as that observed in the absence of the inhibitor. � This is because at the higher concentration the active site will be saturated with substrate which means the inhibitor cannot bind

Non-competitive: (allosteric effect) � These are not treated by the concentration of the substrate.

Non-competitive: (allosteric effect) � These are not treated by the concentration of the substrate. � It inhibits by binding irreversibly to the enzyme but not at the active site. Examples � Cyanide combines with the Iron in the enzymes cytochrome oxidase. 2.

� Km : Non-competitive inhibitors do not interfere with the binding of substrate to

� Km : Non-competitive 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 non-competitive inhibitor. � Vmax: Increasing the concentration of substrate does not overcome non-competitive inhibition. � Non-competitive inhibitors therefore decrease the Vmax of the reaction. � Non-competitive inhibitors therefore simply reduce the amount of active enzyme so they decrease Vmax, but have no effect on Km

Kompetitive Inhibition: Km Increases; no change in Vmax. Non-kompetitive inhibition: No Km change, but

Kompetitive Inhibition: Km Increases; no change in Vmax. Non-kompetitive inhibition: No Km change, but Vmax decreases

Michaelis-Menten Equation

Michaelis-Menten Equation

Glossary � Active site: The region of an enzyme molecule which binds the substrate

Glossary � Active site: The region of an enzyme molecule which binds the substrate and carries out the catalytic reaction � Enzyme : A biological catalyst. Usually a globular protein molecule produced by living organisms that can speed up a specific chemical reaction without itself being destroyed or changed in any way. � K m: (Michaelis constant) The substrate concentration at which an enzyme catalysed reaction proceeds at half the maximum velocity. � V max: (Maximum velocity) The maximum initial velocity of an enzyme catalysed reaction; determined by increasing the substrate [S] until a constant rate of product formation is achieved (i. e. saturating substrate levels).

�A CATALYST is anything that speeds up a chemical reaction that is occurring slowly

�A CATALYST is anything that speeds up a chemical reaction that is occurring slowly

ALKALINE PHOSPHATASE �Is a member of the phosphomonoestrases group. Which are highly specific and

ALKALINE PHOSPHATASE �Is a member of the phosphomonoestrases group. Which are highly specific and act an only one substrate, alkaline phosphatase has a broad substrate specificity and is named alkaline phosphatase because its ph optimum is usually around 9 but other broad spectrum phosphoesterases with ph optima less than 7 are termed acid phosphatases.

Determination ALP in vitro �Alkaline phosphatase activity by following the rate of formation of

Determination ALP in vitro �Alkaline phosphatase activity by following the rate of formation of products. �ALP catalyzes in alkaline medium the transfer of the phosphate group from 4 nitrophenylphosphate to 2 amino 2 methyle propanol, librating 4 nitrophenol �The catalytic concentration is determined from the rate of 4 nitrophenol formation, measured at 400 nm. �The 4 nitrophenol is bright yellow but other reactants and products are colorless in aqueous solution.

�The measurement of ALP activity in vitro is based on artificial substrate p-nitrophenylphosphate. �The

�The measurement of ALP activity in vitro is based on artificial substrate p-nitrophenylphosphate. �The intensity of yellow color in the reaction solution thus indicates the degree to which enzyme has acted upon substrate. (how much enzyme acted on substrate).

Study enzyme kinetics � Enzyme kinetics: the study of the rate at which an

Study enzyme kinetics � Enzyme kinetics: the study of the rate at which an enzyme works. � To examine it, when the substrate available to the enzyme one would do the following: 1. set up the series of tubes containing graded concentrations of substrate 2. At time zero, a fixed amount of the enzyme preparation is added 3. Over the next few minutes, the concentration of product formed, is measured. If the product absorbs light, we can easily do this in a spectrophotometer 4. Early in the run, when the amount of substrate is in substantial excess to the amount of enzyme, the rate we observed is the initial of velocity Vi.

Experiment Facts. . An assay is necessary to study an enzyme The assay is

Experiment Facts. . An assay is necessary to study an enzyme The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product (or otherwise the decrease in substrate conc. )

Reagents and instruments 18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to

Reagents and instruments 18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to use) Blood serum 5 N Na. OH solution

Procedure Take 18 clean plastic tubes and label them from 1 to 18. Another

Procedure Take 18 clean plastic tubes and label them from 1 to 18. Another tube will be used as a blank. This will contain all the reagents except of the enzyme. Make the substrate and buffer concentrations as described in the given table (make sure to keep the total volume of all tubes stable at 1. 9 ml). Transfer 100 µl of serum to each tube. Mix substrate and serum solutions and incubate at 37 c for 50 seconds. Add 0. 5 ml of 5 N Na. OH in each tube to stop the reaction. Read the absorbance at 400 nm. Plot reaction rate (Vi) on Y axis against substrate concentration [S] on X axis.

Tube Buffer Substrate Enzyme 5 N Na. OH Abs. at μl μl 400 nm

Tube Buffer Substrate Enzyme 5 N Na. OH Abs. at μl μl 400 nm 1 100 1800 100 500 2 200 1700 100 500 3 300 1600 100 500 4 400 1500 100 5 500 1400 100 500 6 600 1300 100 500 7 700 1200 100 500 8 800 1100 500 9 900 100 500 10 1000 900 100 500 11 1100 800 100 500 12 1200 700 100 500 13 1300 600 100 500 14 1400 500 15 1500 400 100 500 16 1600 300 100 500 17 1700 200 100 500 18 1800 100 500