Experiment4 Effects of substrate concentration on enzyme activity ——determination of Km of alkaline phosphatase ）

Objective: To understand the effect of substrate concentration on the velocity of enzymed- catalyzed reaction and to learn the principle and method for determination of the Michaelis constant (Km).

Principle: In the condition of constant temperature, PH and concentration of enzyme, the concentration of substrate has more effect on the velocity of enzymed-catalyzed reaction. In normal condition, at very low concentration of substrate, as the concentration of substrate is increased, the velocity of reaction is increased quickly; as the concentration of substrate is increased continuously, the increase of velocity starts slow down; as the concentration of substrate is increased to some level, the velocity approaches to a maximum velocity (Vmax).

The relation between the concentration of substrate and the velocity can be expressed with the formulation of the Michaelis-Menten equation:

( 一 ) a plot of V versus [S] From the formulation of the Michaelis-Menten equation, an important numerical relationship emerges in the special case when V is exactly one- half Vmax, Km=[S]. Thus, Km is equal to the concentration of substrate at which the reaction rate is half its maximal velocity (fig.6-1)

Fig.6-1 a plot of V versus [S]

（二） a plot of 1/v versus 1/[s] The Michaelis-Menten equation can be algebraically transformed into Lineweaver-Burk equation: A plot of 1/v versus 1/[s] yields a straight line. This line will have a slope of Km/Vm, an intercept of 1/Km on the 1/[s] axis.

Fig. 6-2 a plot of 1/v versus 1/[s]

AKP OH — C 6 H 5 PO 4 Na 2 + H 2 O C 6 H 5 OH + Na 2 HPO 4 C 6 H 5 OH + 4-amino antipyrine quinine derivant OH — potassium ferricyanide

Procedure: 1. Take 10 clean and dry small test tubes and manipulate according to the following table. Note: take reactant acculately and mix it completely.

Table Effects of substrate concentration on enzyme activity The number of tubes standard tube Phenol standard Solution(0.1mg/L) (ml) mg/L Substrate solution (ml) H 2 O (ml) mol/L, PH10 carbonic acid buffer (ml) 37 o C water bath for 5 minutes AKP enzymatic solution (ml)

Note: After AKP solution was added to each tube, reckon by time quickly. Mix it softly and keep in 37 o C water bath for 15minutes. 2. When 15 minutes is up, add 1ml 0.5mol/L NaOH solution quickly to stop reaction. 3. Add 3g/L 4-amino antipyrine 1.0ml and 5g/L potassium ferricyanide 2.0ml to each tube, mix it completely, place them for 10 minutes. Then examine the OD510 respectively used No.1 tube as the blank tube, regulate its OD value to 0.00 and write down the OD510 of each tube.

4. A plot of 1/V versus 1/[s] yields a straight line, an intercept on the 1/[s] axis is -1/Km, then calculate the Km. Results and calculate 1. calculate the [s] of each tube. 2. fill each OD510 value in following table. 3.calculate the velocity of reaction in each tube according to the OD510 value. 4. draw a plot of 1/v versus 1/[s].

The number of tubes standard tube [s] 1/[s] OD 510 1/ OD 510 v(mg phenyl/min) 1/v

Question: 1.Which are keys in the processing of determination Km ? 2. We can also draw a plot of 1/ OD510 versus 1/[s], an intercept on the 1/[s] axis is 1/Km. Why?