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May 28, 2016

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describes the role of pH on enzyme activity

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38 views

describes the role of pH on enzyme activity

© All Rights Reserved

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You are on page 1of 5

ph.mcd

Effect of pH.

Instructor: Nam Sun Wang

Mechanism #1 -- Acid Enzyme. Substrate can be protonated, and only the protonated form can

combine with an enzyme to form an active complex.

S + H+ (inactive)

K2

Km

k2

+

+

SH + E ESH E + PH+

Derivation of Reaction Rate Expression with Equilibrium Assumption .

Given

v k 2 . ESH

E 0 E ESH

3. Equilibrium Assumption:

Km

1. dp/dt=rate=v

SH. E

ESH

K2

S. H

SH

We have 4 eqns to solve for 4 unknowns -- E, ESH , SH, and v. Find the analytical expression (via

|Math|SmartMath|)

E 0 . K m.

S. H.

Find( E , ESH , SH , v )

K2

K m. K 2

S. H

E0

K m. K 2

S. H

H

S.

K2

k 2 . S. H.

E0

K m. K 2

S. H

k 2 . E 0 . S. H

k 2. E 0. S

v m. S

v

where

K m. K 2 S. H

K2

K mapp S

K m.

S

H

v m k 2. E 0

K mapp K m.

K2

H

ph.mcd

Mechanism #2 -- Alkaline Enzyme. Substrate can give off a proton, and only the ionized form can

combine with an enzyme to form an active complex.

Km

k2

S- + E ES- E + P+

H+

K 1

S (inactive)

Derivation of Reaction Rate Expression with Equilibrium Assumption .

Given

1. dp/dt=rate=v

2. Conservation of enzyme species

3. Equilibrium Assumption:

v k 2 . ES

E 0 E ES

S i. E

Km

ES

K1

S i. H

S

We have 4 eqns to solve for 4 unknowns -- E, ES, Si, and v. Find the analytical expression (via

|Math|SmartMath|)

H

E 0 . K m.

K 1 . S.

Find E , ES , S i , v

K m. H

K 1. S

E0

K m. H

K 1.

k 2 . K 1 . S.

K 1. S

S

H

E0

K m. H

K 1. S

k 2 . E 0 . S. K 1

k 2. E 0. S

v m. S

v

where

K m. H K 1 . S

H

K

S

mapp

K m.

S

K1

v m k 2. E 0

H

K mapp K m.

K1

ph.mcd

Mechanism #3 -- Ionizing Enzyme . Enzyme can be ionized, and only one form is active.

E- + H+ (inactive)

K2

Km

k2

EH + S EHS EH + P

+

H+

K1

EH2 + (inactive)

Derivation of Reaction Rate Expression with Equilibrium Assumption .

Given

v k 2 . EHS

E 0 E EH

3. Equilibrium Assumption:

K m'

1. dp/dt=rate=v

EH 2

EH. S

EHS

EH. H

K1

EHS

K2

EH 2

E. H

EH

We have 5 eqns to solve for 5 unknowns -- E, EH, EH2 , EHS, and v. Find the analytical expression

(via |Math|SmartMath|)

K 2 . K m'. K 1 .

K m'. K 1 . E 0 .

E0

K 1 . K 2 . K m'

K 1 . K m'. H

K 1 . H. S

2

K m'. H

K 1 . H. S

2

K m'. H

H

K 1 . K 2 . K m'

K 1 . K m'. H

2

E 0 . K m'.

Find E , EH , EH 2 , EHS , v

K 1 . E 0 . H.

H

K 1 . K 2 . K m'

K 1 . K m'. H

K 1 . H. S

2

K m'. H

S

K 1 . K 2 . K m'

k 2 . K 1 . E 0 . H.

K 1 . K m'. H

2

K m'. H

K 1 . H. S

S

K 1 . K 2 . K m'

K 1 . K m'. H

K 1 . H. S

2

K m'. H

Thus, the last row is the analytical expression for v. We can further rearrange it by following these

steps: copy the denominator, divide by HK 1 , mark the whole expression and choose

|Symbolic|Expand Expression|, mark "Km" and choose |Symbolic|Collect on Subexpression| to

collect Km terms, finally place the resulting denominator back into the expression for v.

k 2 . E 0 . S. H. K 1

v

K m. K 2 . K 1

K m. H. K 1

2

K m. H

k 2. E 0. S

S. H. K 1 K . 1 . K

m

2

H

1 .

H

K1

ph.mcd

Thus, the above form is transformed into the Michaelis-Menten form by defining:

1

K mapp K m. . K 2

H

v m k 2. E 0

1 .

H

K1

v m. s

v( s )

K mapp

pH

K m. 10

pK 2

10

pK 1

pH

Optimum pH. The rate goes through a maximum when dv/dpH=0. Let's find where this maximum

lies. Here, we mark "H" in the following equation and choose |Symbolic|Differentiate on Variable|.

v m. s

v

1

1 .

K m. . K 2 1

H

s

H

K1

vm

1

.K . 1 .K

0 s.

m

2

2

2

K1

1

1 .

H

K m. . K 2 1

H

s

H

K1

Choose |Symbolic|Simplify| yields:

2

K 1. K 2

0 s. v m. K 1 . K m.

K m. K 2 . K 1

2

K m. H

K m. H. K 1

s. K 1 . H

Since there is only one equation, mark "H" in the above equation and choose |Symbolic|Solve for

Variable| yields:

K 1. K 2

K 1. K 2

Since the H concentration must be non-negative, we choose the first solution.

H max

K 1. K 2

To find the maximum v at optimum pH, copy "K1 K2 " into the clipboard, mark "H max" in the following

equation for vopt, and choose |Symbolic|Substitute for Variable|.

v m. s

v opt

1 .

1 .

K m.

K2 1

H max

s

H max

K1

v m. s

v opt

K m. 2 .

K2

K1

v m. s

pK 1

s K m. 2 . 10

pK 2

2

Plot of pH dependence v m

Km

Acid enzyme:

pK 1

v acid( s , pH )

Alkaline enzyme:

ph.mcd

K m. 10

K m. 10

pH

pK 1

pH

v m. s

K m. 10

pH

pK 2

pK 1

10

pH

v m. s

v opt( s )

pK 1

K m. 2 . 10

pH

pK 2

v m. s

v( s , pH )

v m. s

v alkaline( s , pH )

Ionizing enzyme:

pK 2

pK 2

2

4 , 4.1 .. 10

1

Enzyme Activity

pK 1

pK 2

v acid ( 1 , pH )

v alkaline ( 1 , pH )

0.5

v( 1 , pH )

v opt( 1 )

0

4

5.5

8.5

10

pH

Acid Enzyme

Alkaline Enzyme

Ionizing Enzyme

Acid enzymes have high activities at acidic conditions. Whereas, alkaline enzymes have high

activities at alkaline conditions. For these enzymes, the enzyme activity drops to half of the

maximum level at pH=pK,. Ionizing enzymes exhibit a maximum at a pH halfway between pK 1 and

pK2 . The smaller the difference between pK 1 and pK2 , the narrower the active range. Finally, all of

these mechanisms give rise to a change in the apparent K m value; thus, competitive inhibition.

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