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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 mole of substrate into product per minute at 25C.(1 mole = 1 x 10-6moles) Katal : is defined as the number of moles of substrate transformed into product per second at 25C.

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Specific activity: It is defined as the number of enzyme units per milligram of protein (mole/min/mg of protein)(mole.min-1.mg of protein-1)This is valuable during enzyme purification.As enzyme become pure, specific activity increases.

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Turnover number
Turnover number: It is the number of moles of substrate transformed per minute per mole of enzyme (Units per mole of active site or catalytic center under optimum conditions). This tells how many S molecules are converted to product by each enzyme molecule. It tells us how fast an enzyme work or turnover S into P. e.g. for catalase:turnover number is 5 x 106for -amylase it is 1.9 x 104This indicates that catalase is ~ 250 times more active than amylase
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Enzyme Kinetics
-The study of reaction rates and how they change in response to changes in experimental parameters in known as Kinetics. -Kinetics is that branch of enzymology that deals with the factors that affect the rate of enzyme catalysed reactions.

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Factors affecting Enzyme Reaction Velocity

(i)Enzyme concentration. (ii)Substrate concentration. (iii)Temperature (iv)pH. (v)Activators. (vi)Inhibitors

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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]; v = k [E] As E increases rate of reaction increases in a linear manner.
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Effect of Enzyme concentration on rate of Enzymic reaction

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Effect of Enzyme concentration on rate of Enzymic reaction

some deviations occur: (a) upward curve (b) downward curver [E]

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Upward curve
v against [E] curve:

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Reasons for upward curve

1.Presence of highly toxic impurity in the reaction in the reaction mixture (not in E solution).So when E is in small amount it is inhibited, but as its concentration is increased, it overcomes the toxic impurity and rate increases.[E]v 2.Presence of a dissociable activator or coenzyme in the enzyme preparation. (e.g. some proteases) 3.Some E become active as they aggregate at high concentration. (e.g. 6-phosphofructokinase)
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Downward Curve

This is more common. As E concentration is increased beyond a certain point, the rate decreases.

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Downward curve for v against[E]

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Reasons for downward curve

1.limitation in the capacity of the method of estimation. This is not a true decrease, but occurs as the assay method cannot give higher reading. (e.g. in spectrophotometer the maximum O.D. is 2.0). 2.The coenzyme may be limited and as the E remains as ApoE and loses activity. 3.Substrate may be used up. 4.Presence of a reversible inhibitor in the enzyme preparation. As E concentration increases, I increases and inhibits E.
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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 r[S]. A straight line is obtained. As S concentration is increased a mixed order reaction is obtained and the curve reaction is obtained. As S is increased further the rate does not change and becomes constant. This is because E active sites are all filled and E is saturated with S. At this point the velocity is equal to Maximum Velocity(Vmax). The S concentration at half Vmax (Vmax/2) is called Michaelis Constant(Km). This is a constant for an E and a specific substrate. It gives the affinity between E and S. High Km indicates low affinity Low Km indicates high affinity.

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Effect of Substrate Concentration on the rate of E catalysed reaction

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V against [S] curve

The E which give hyperbolic curve with S obey MiichaelisMenten kinetics. However, some E do not obey Michaelis-Menten kinetics and do not give a hyperbolic curve, but give a Sigmoid curve. These are allosteric enzymes. These are regulatory enzymes and have a quarternary structure.

Sigmoid curve indicates cooperative effect

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Allosteric Enzymes
Allosteric Enzymes have multiple binding sites: Active sites: binds S and converts to P. Modulatory site:binds S and other modulatory molecules and this binding affects the activity of active site. Modulators may be:+veModulators activity -veModulators activity.
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Effect of Temperature on E catalysed reactions:

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Effect of Temperature on E catalysed reactions:

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

When E actvity is measured at several pH values, optimal activity is generally observed between pH values of 5-9. However, some E such as pepsin have low pH optimum ( 2.0) which others have high pH optimum (e.g. Alkaline Phosphatase(pH ~ 9.5).

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pH activity curve
The shape of pH activity curve is determined by the following: (i)E is denaturedat high or low pH. (ii)Alteration in the charge state of the E or S or both. For E pH can affect activity by changing the structure or by changing the charge on a.a. which are functional in S binding or catalysis e.g. Enz-+ SH+Enz.SH # At Low pH:Enzyme is protonated and loses its negative charge, Enz-+ SH+ Enz-SH # At high pH :The substrate loses its proton and positive charge SH+S-+ H+ So Enz-+ S-No reaction

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Effect of pH on enzyme catalysed reactions

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(v) Effect of Inhibitors on rate of E catalysed reaction:

Inhibitors are substances that combine with E and decrease its activity. Presence of I decreases the rate of E catalysed reaction. Inhibitors may be: i. Irreversible inhibitor ii. Reversible inhibitors

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Irreversible inhibitor
E + IEI -This inhibitor cannot be removed by dialysis or other means: -Inhibition increases with time. Examples of irreversible inhibitors CN inhibits xanthine oxidase. Nerve gas inhibits cholinesterase. Iodoacetamide, heavy metal ions (Hg++),oxidising agents.
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Reversible inhibitor
E + IEI -The reaction is reversible and the I can be removed by dialysis or other means. Reversible inhibitors are of three types: 1. Competitive 2.Noncompetitive 3.Uncompetitive
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Reversible inhibitor
Examples of reversible inhibitors: Inhibition of succinate dehydrogenase by malonate. Inhibition of methanol dehydrogenase by ethanol.

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(v)Effect of Activators on rate of E catalysed reactions.

Some E require activators to increase the rate of reaction.
Activators cause activation of E-catalysed reaction by either altering the velocity of the reaction or the equilibrium reached or both. e.g.: Essential activators: Essential for the reaction to proceed. These are recognised as substrate that is not changed in the reaction e.g. metal ion such as Mg++for kinases. Non essential activators: Activator may act to promote a reaction which is capable of proceeding at a appreciable rate in the absence of activator.
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