The enzyme alcohol dehydrogenase catalyzes the oxidation of alcohol by NAD+ to g

Question

The enzyme alcohol dehydrogenase catalyzes the oxidation of alcohol by NAD+ to give acetaldehyde and NADH. NADH has an absorption maximum at 340 nm with an extinction coefficient of 6220 M-1s-1, whereas NAD+ and the other reactants do not absorb significantly at 340 nm. Consequently, the oxidation reaction can be conveniently monitored by following the increase in absorbance at 340 nm.

A.) What is the rate of production of NADH if it is observed that the rate of absorbance increase in a 1 cm path length cell is 0.03 1/min?

B.) When an excess of enzyme is added to NADH, the absorbance at 340 nm decreases by 10%. What is the extinction coefficient of NADH at 340 nm when it is bound to the enzyme?

C.) The extinction coefficient can be used to determine the binding constant for the association of NADH and the enzyme. The following data were obtained for the difference absorbance when NADH is added to 10 M enzyme in a 1 cm path length cell.

D.) Graphically determine the binding constant from the data in the table.

Explanation / Answer

1) A = E*C*L

E= molar absoptivity coefficient; C= concentration; L=path length

Maximum absobance (1) at 340 nm; extinction coefficient of 6220 M-1s-1

Rate of absorbance increase = 0.031/min

Lets say there is an increase of x then, 1-x/1*100=0.031

100-100x=0.031

-100x= 0.031-100=-99.97

100x=99.97

x=0.99

Now, new C will be

0.99= 6220*M-1s-1*1cm*C

=1.5x10-4

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