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Enzymology [Compatibility Mode]

Enzymology [Compatibility Mode]

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Published by: Toby FitzSimons on Apr 30, 2012
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12/04/1430

Enzyme Kinetics
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Enzyme activity
Enzyme activity:It is defined as the amount of enzyme that will convert a certain amount of S to P in a specified period of time under conditions of constant temperature and pH. •The international enzyme commission (IEC) have adapted a standard unit of enzyme activity called The International Unit (IU). •The International Unit (IU) is defined as the amount of enzyme that can convert one μmole of substrate into product per minute at 25°C.(1 →μmole = 1 x 10-6moles) Katal : is defined as the number of moles of substrate transformed into product per second at 25°C.

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Dr.Saba Abdi

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g. It tells us how fast an enzyme work or turnover S into P.12/04/1430 .Saba Abdi Turnover number Turnover number: It is the number of moles of substrate transformed per minute per mole of enzyme (Units per μmole of active site or catalytic center under optimum conditions). 3 Dr. specific activity increases. Specific activity: It is defined as the number of enzyme units per milligram of protein (μmole/min/mg of protein)(μmole. This tells how many S molecules are converted to product by each enzyme molecule.min-1.9 x 104This indicates that catalase is ~ 250 times more active than amylase 4 Dr. for catalase:turnover number is 5 x 106for α-amylase →it is 1.Saba Abdi 2 .As enzyme become pure.mg of protein-1)This is valuable during enzyme purification. e.

Saba Abdi Factors affecting Enzyme Reaction Velocity (i)Enzyme concentration. (iii)Temperature (iv)pH. (ii)Substrate concentration.12/04/1430 Enzyme Kinetics -The study of reaction rates and how they change in response to changes in experimental parameters in known as Kinetics. (v)Activators.Saba Abdi 3 . (vi)Inhibitors 6 Dr. 5 Dr. -Kinetics is that branch of enzymology that deals with the factors that affect the rate of enzyme catalysed reactions.

v = k [E] As E increases rate of reaction increases in a linear manner.Saba Abdi Effect of Enzyme concentration on rate of Enzymic reaction 8 Dr. 7 Dr.12/04/1430 Effect of Enzyme concentration on rate of Enzymic reaction The rate of E catalysedreaction is proportional to the Enzyme concentration(provided S is saturating E) v ∝[E].Saba Abdi 4 .

Saba Abdi Upward curve v against [E] curve: 10 Dr.Saba Abdi 5 .12/04/1430 Effect of Enzyme concentration on rate of Enzymic reaction some deviations occur: (a) upward curve (b) downward curver α[E] 9 Dr.

12 Dr. (e.g.Presence of highly toxic impurity in the reaction in the reaction mixture (not in E solution).g. •As E concentration is increased beyond a certain point. (e.Saba Abdi Downward Curve •This is more common.So when E is in small amount it is inhibited. the rate decreases.Some E become active as they aggregate at high concentration. 6-phosphofructokinase) 11 Dr.[E]v 2. it overcomes the toxic impurity and ∴rate increases. some proteases) 3. but as its concentration is increased.Saba Abdi 6 .12/04/1430 Reasons for upward curve 1.Presence of a dissociable activator or coenzyme in the enzyme preparation.

in spectrophotometer the maximum O.Saba Abdi Reasons for downward curve 1.0). 4.Presence of a reversible inhibitor in the enzyme preparation. 14 Dr. This is not a true decrease.limitation in the capacity of the method of estimation. 3. I increases and inhibits E.D. (e. is 2.g. 2.Saba Abdi 7 .12/04/1430 Downward curve for v against[E] 13 Dr. As E concentration increases.Substrate may be used up.The coenzyme may be limited and as the E remains as ApoE and ∴loses activity. but occurs as the assay method cannot give higher reading.

At this point the velocity is equal to Maximum Velocity(Vmax). This is because E active sites are all filled and E is saturated with S.Saba Abdi 8 . High Km indicates low affinity Low Km indicates high affinity. 15 Dr. This is a constant for an E and a specific substrate. A straight line is obtained. •As S is increased further the rate does not change and becomes constant. It gives the affinity between E and S.12/04/1430 Effect of Substrate Concentration on the rate of E catalysed reaction S most important factor in determining velocity of E reaaction •At Low S concentration rate of reaction is low and rα[S]. As S concentration is increased a mixed order reaction is obtained and the curve reaction is obtained. •The S concentration at half Vmax (Vmax/2) is called Michaelis Constant(Km).Saba Abdi Effect of Substrate Concentration on the rate of E catalysed reaction 16 Dr.

•Modulatory site:binds S and other modulatory molecules and this binding affects the activity of active site. • Modulators may be:+veModulators →↑activity -veModulators →↓activity. but give a Sigmoid curve.Saba Abdi 9 .Saba Abdi Allosteric Enzymes Allosteric Enzymes have multiple binding sites: •Active sites: binds S and converts to P. •However. Sigmoid curve indicates cooperative effect 17 Dr.12/04/1430 V against [S] curve The E which give hyperbolic curve with S obey MiichaelisMenten kinetics. These are allosteric enzymes. 18 Dr. some E do not obey Michaelis-Menten kinetics and do not give a hyperbolic curve. These are regulatory enzymes and have a quarternary structure.

Some E in bacteria which survive in hot springs have high optimal temperature 20 Dr.Saba Abdi Effect of Temperature on E catalysed reactions: At very low temperature e. •As temperature is increased rate of reaction increases. optimal temperature are at or slightly above those of the cell in which the E occurs.Saba Abdi 10 . •This occurs as the kinetic energy of the molecules increases.12/04/1430 Effect of Temperature on E catalysed reactions: 19 Dr. This is Q10 or Temperature Coefficient. O°C the rate of reaction may be almost zero. •Beyond this temperature. •For most E.g. the rate decreases sharply. •For every 10°C rise of temperature the rate is doubled. This occurs as the enzyme is denatured and the catalytic activity is lost. •But this occurs only upto a specific temperature which is known as Optimum temperature.

optimal activity is generally observed between pH values of 5-9. (ii)Alteration in the charge state of the E or S or both. 21 Dr.g.g. Enz-+ SH+ →Enz-SH # At high pH :The substrate loses its proton and ∴positive charge SH+→S-+ H+ So Enz-+ S-→No reaction 22 Dr. Alkaline Phosphatase(pH ~ 9.Saba Abdi 11 .a. When E actvity is measured at several pH values.12/04/1430 Effect of pH on E catalysed reaction.5). •For E pH can affect activity by changing the structure or by changing the charge on a. However.Saba Abdi pH activity curve The shape of pH activity curve is determined by the following: (i)E is denaturedat high or low pH. Enz-+ SH+→Enz. which are functional in S binding or catalysis e.0) which others have high pH optimum (e.SH # At Low pH:Enzyme is protonated and loses its negative charge. some E such as pepsin have low pH optimum ( 2.

Inhibitors may be: i. Irreversible inhibitor ii. •Presence of I decreases the rate of E catalysed reaction. Reversible inhibitors 24 Dr.12/04/1430 Effect of pH on enzyme catalysed reactions 23 Dr.Saba Abdi 12 .Saba Abdi (v) Effect of Inhibitors on rate of E catalysed reaction: •Inhibitors are substances that combine with E and decrease its activity.

12/04/1430 Irreversible inhibitor E + I→EI -This inhibitor cannot be removed by dialysis or other means: -Inhibition increases with time. •Iodoacetamide. Competitive 2.Saba Abdi Reversible inhibitor E + I↔EI -The reaction is reversible and the I can be removed by dialysis or other means. 25 Dr. heavy metal ions (Hg++). Reversible inhibitors are of three types: 1. Examples of irreversible inhibitors •CN inhibits xanthine oxidase. •Nerve gas inhibits cholinesterase.Uncompetitive 26 Dr.Noncompetitive 3.oxidising agents.Saba Abdi 13 .

g.g. •Inhibition of methanol dehydrogenase by ethanol.: Essential activators: Essential for the reaction to proceed.Saba Abdi 14 . Non essential activators: Activator may act to promote a reaction which is capable of proceeding at a appreciable rate in the absence of activator. These are recognised as substrate that is not changed in the reaction e. •Some E require activators to increase the rate of reaction.12/04/1430 Reversible inhibitor Examples of reversible inhibitors: •Inhibition of succinate dehydrogenase by malonate. 27 Dr. e.Saba Abdi (v)Effect of Activators on rate of E catalysed reactions. 28 Dr. •Activators cause activation of E-catalysed reaction by either altering the velocity of the reaction or the equilibrium reached or both. metal ion such as Mg++for kinases.

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