A 1 month old baby is failing to thrive, with poor feeding and growth, and frequent irritability. After becoming ill with a severe cold, he is admitted to hospital with abdominal tenderness and yellowing of the whites of the eyes, and is diagnosed with jaundice. Clinical investigation reveals elevated serum bilirubin, splenomegaly, and a low level of haemoglobin.   Whole blood extract containing 200mg of red blood cells were resuspended in 1mL saline stabilised with 1,8-beta-mercaptoethanol and EDTA. The activity of “enzyme X” was measured in the sample by adding 100µL of this suspension to 0.9 mL of Tris-HCl EDTA buffer (pH 8.0) containing 0.1M MgCl2, 2.0mM NADH, 50mM phosphoenolpyruvate (PEP), 30mM ADP and lactate dehydrogenase at 60units/mL.   The decrease in absorbance at 340nm was followed over a period of 10 minutes and compared with a blank sample containing all components except the ADP. The results are shown in the table below:   Time/min Absorbance   With ADP Without ADP 0 0.53 0.092 2 0.44 0.087 4 0.34 0.082 6 0.23 0.062 8 0.12 0.03 10 0 0   From the constituents of the assay, identify “enzyme X” and write out the reaction it catalyses, and the coupling reaction. Why is the coupling reaction needed?

Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
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A 1 month old baby is failing to thrive, with poor feeding and growth, and frequent irritability. After becoming ill with a severe cold, he is admitted to hospital with abdominal tenderness and yellowing of the whites of the eyes, and is diagnosed with jaundice. Clinical investigation reveals elevated serum bilirubin, splenomegaly, and a low level of haemoglobin.

 

Whole blood extract containing 200mg of red blood cells were resuspended in 1mL saline stabilised with 1,8-beta-mercaptoethanol and EDTA. The activity of “enzyme X” was measured in the sample by adding 100µL of this suspension to 0.9 mL of Tris-HCl EDTA buffer (pH 8.0) containing 0.1M MgCl2, 2.0mM NADH, 50mM phosphoenolpyruvate (PEP), 30mM ADP and lactate dehydrogenase at 60units/mL.

 

The decrease in absorbance at 340nm was followed over a period of 10 minutes and compared with a blank sample containing all components except the ADP. The results are shown in the table below:

 

Time/min

Absorbance

 

With ADP

Without ADP

0

0.53

0.092

2

0.44

0.087

4

0.34

0.082

6

0.23

0.062

8

0.12

0.03

10

0

0

 

From the constituents of the assay, identify “enzyme X” and write out the reaction it catalyses, and the coupling reaction. Why is the coupling reaction needed? 

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