Enzymology [Compatibility Mode]

8/2/2019 Enzymology [Compatibility Mode]

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Enzyme Kinetics

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Enzyme activity

Enzyme activity:It is defined as the amount of enzyme thatwill 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 astandard unit of enzyme activity called The InternationalUnit (IU).

The International Unit (IU) is defined as the amount ofenzyme that can convert one mole of substrate intoproduct per minute at 25C.(1 mole = 1 x 10-6moles)

Katal : is defined as the number of moles of substratetransformed 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: It is the number of moles of substratetransformed per minute per mole of enzyme (Units permole of active site or catalytic center under optimumconditions).

This tells how many S molecules are converted to productby 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 -amylaseit is 1.9 x 104This indicates that catalase is ~ 250times more active than amylase

Turnover number

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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 proportionalto the Enzyme concentration(provided S issaturating E)

v [E]; v = k [E]

As E increases rate of reaction increases in alinear manner.

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Effect of Enzyme concentration on rate of Enzymicreaction

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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 curvev against [E] curve:

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Reasons for upward curve

1.Presence of highly toxic impurity in the reaction inthe reaction mixture (not in E solution).So when Eis in small amount it is inhibited, but as itsconcentration is increased, it overcomes the toxicimpurity and rate increases.[E]v

2.Presence of a dissociable activator or coenzyme inthe enzyme preparation.

(e.g. some proteases)

3.Some E become active as they aggregate at highconcentration.

(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 assaymethod cannot give higher reading. (e.g. inspectrophotometer the maximum O.D. is 2.0).

2.The coenzyme may be limited and as the E remains asApoE and loses activity.

3.Substrate may be used up.

4.Presence of a reversible inhibitor in the enzymepreparation.

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 lineis obtained. As S concentration is increased a mixed order reaction isobtained and the curve reaction is obtained.

As S is increased further the rate does not change and becomesconstant. This is because E active sites are all filled and E is saturatedwith S. At this point the velocity is equal to Maximum Velocity(Vmax).

The S concentration at half Vmax (Vmax/2) is called MichaelisConstant(Km). This is a constant for an E and a specific substrate. Itgives the affinity between E and S.

High Km indicates low affinityLow 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 Miichaelis-Menten kinetics.

However, some E do not obey Michaelis-Menten kineticsand do not give a hyperbolic curve, but give a Sigmoidcurve. These are allosteric enzymes. These areregulatory 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 almostzero.

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 orTemperature Coefficient.

But this occurs only upto a specific temperature which is known asOptimum temperature.

Beyond this temperature, the rate decreases sharply. This occurs asthe enzyme is denatured and the catalytic activity is lost.

For most E, optimal temperature are at or slightly above those of thecell in which the E occurs. Some E in bacteria which survive in hotsprings 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 changingthe charge on a.a. which are functional in S binding or catalysis

e.g. Enz-+ SH+Enz.SH

loses its negative charge,andprotonatedis:EnzymepHLowAt#

Enz-+ SH+ Enz-SH

positive chargesubstrate loses its proton andThe:high pHAt#SH+S-+ H+

So Enz-+ S-No reaction

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Effect of pH on enzyme catalysedreactions

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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 inhibitorii. 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

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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 for the reaction to proceed. TheseactivatorsEssential

are recognised as substrate that is not changed in the reaction

e.g. metal ion such as Mg++for kinases.

: Activator may act to promote a reactionessential activatorsNon

which is capable of proceeding at a appreciable rate in the

absence of activator.

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Enzymology [Compatibility Mode]