1

2

ENZYMESo Enzyme reaction:

E + S ↔ ES → E + PE + S ↔ ES → E + P

Whereas: E: Enzyme, S: Substrate, ES: Enzyme-Substrate complex, P: Product

3

ENZYMES DECREASE ACTIVATION ENERGY

A chemical reaction goes through a transition state with a higher G than either S of P

Enzymes facilitate the formation of the transition state by decreasing G‡

4

Factors influencing an enzymatic reaction

1. Substrate concentration2. Temperature3. Inhibitors4. Activators5. pH

5

1. SUBSTRATE CONCENTRATE At low values of [S],

the initial velocity,Vi, rises almost linearly with increasing [S].

But as [S] increases, the gains in Vi level off (forming a rectangular hyperbola).

The asymptote represents the maximum velocity of the reaction, designated Vmax

6

The substrate concentration that produces a Vi that is one-half of Vmax is designated the Michaelis-Menten constant, Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affinity (so the lower the concentration of substrate needed to achieve a given rate).

7

ALLOSTERIC EFFECTORS• Noncovalently bind

and regulate the enzyme.

• The effector may be stimulatory or inhibitory.

• The substrate and effector usually occupy different specific binding sites.

8

9

10

BACKGROUND

11The reaction catalyzed by alkaline Phosphatase

Function R-PO4 + H2O → R-OH + H3PO4

substrate product product

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.)

12

REAGENTS AND INSTRUMENTS

18 labeled plastic tubes Micropipette Spectrophotometer ALP enzyme kit (ready to use) Blood serum 5 N NaOH solution

13

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

37c for 50 seconds. Add 0.5 ml of 5 N NaOH 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.14

Tube Bufferμl

Substrateμl

Enzymeμl

5 N NaOHμl

Abs. at 400nm

1 100 1800 100 500

2 200 1700 100 500

3 300 1600 100 500

4 400 1500 100 500

5 500 1400 100 500

6 600 1300 100 500

7 700 1200 100 500

8 800 1100 100 500

9 900 1000 100 500

10 1000 900 100 500

11 1100 800 100 500

12 1200 700 100 500

13 1300 600 100 500

14 1400 500 100 500

15 1500 400 100 500

16 1600 300 100 500

17 1700 200 100 500

18 1800 100 100 500

15