4a-f 4. In addition to the Class I liver ADH above, there are five other classes

Question

4a-f

4. In addition to the Class I liver ADH above, there are five other classes of human ADH. All are encoded as a group on chromosome 4q22. The Class IV isozyme is expressed in epithelial tissues, such as the mucosal lining of the stomach and upper intestines. There is evidence that it is involved in the oxidation of retinol to retinal, the precursor of retinoic acid, the form of vitamin A that serves as a signaling agent in regulation of gene expression involved in growth, development, and epithelial maintenance. The active isomers of retinoic acid, which bind to nuclear receptor proteins are HC CH CHy HyC all-trans-retinoic acid 9 cis-retinoic acid Class IV ADH follows a Bi Bi single displacement reaction mechanism with turnover numbers of 67 min (all-trans-retinol), 475 min (9-cis-retinol), and 2570 min (ethanol) Quenching of the florescence of a tryptophan residue has been used to monitor binding of substrates to Class IV ADH yielding the data in the Table below. The binding reactions contained 56 nM enzyme in O. 1 M sodium phosphate, pH 7.5, 2% (v/v) acetonitrile, and 0.02% (v/v) Tween-80 and were run at 25°C NAD ethanol all-trans-retinol cis-9-retinol 0.0080 0.0151 0.0269 0.0512 1000 0.0731 50 100 200 500 200 500 1000 0.00298 2000 0.00582 5000 0.01362 10 20 30 40 50 0.0512 0.0731 0.0853 0.0931 0.0985 10 20 30 40 50 0.0278 0.0457 0.0582 0.0674 0.0744 0.00061 0.00151 a. Use Excel or similar software to make three Scatchard plots-one for NAD+, one for ethanol, and one for the retinols -to answer the following questions. Include the coordinate values in a b. Calculate the Ks values and number of binding sites n for NAD", ethanol, and each of the retinol c. Do the results indicate that Class IV ADH follows an ordered rapid equilibrium reaction d. Which retinol binds better? How many fold better? table and show the trendline equations on the plot substrates mechanism with regard to substrates like the Class I ADH in question 2? State your rationale e. Calculate enzyme efficiencies with the ethanol and retinol substrates. Do the values support the designation of Class IV ADH as a retinol dehydrogenase rather than alcohol dehydrogenase? Justify your answer f. Why are 0.1 M sodium phosphate and 0.02% Tween-80 included in the binding reactions?

Explanation / Answer

4a.The question can be answered using Scatcherd Plot. The steps are:

1. Calculate [S]b/[S]f for each ligands and from the given table.

2. Draw a graph plotting [S]b on X- and [S]b/[S]f on Y-axis.

3. Draw a trend line using excel itself. While drawing the trend line also check the equation box. The equation will have a Y=mX+C form. The m value is the slope.

4B. The slope is -1/Kd or Ks for that particular ligand.

To calculate the number of binding sites, first calculate the [E]/[S]b values. Take 56nM (given) as the [E].

Draw another graph plotting [E]/[S]b on X-axis and [S]b/[S]f on Y-axis.

The intercept on X-axis will give you the number of binding sites. If the number is not a whole number, round it to nearest whole value.

What I found is :

Number of Binding sites for NAD+ = 2

for Ethanol= 1

for All trans RA and 9 cis RA =2.3;

Hope this is clear.

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