Tyrosine phenol lyase catalyzes the reversible conversion of tyrosine to phenol,
ID: 575840 • Letter: T
Question
Tyrosine phenol lyase catalyzes the reversible conversion of tyrosine to phenol, pyruvate and ammonium. The enzyme uses pyridoxal 5-phosphate (PLP) as a cofactor. The enzyme catalyzes similar reactions as well, for example the reversible conversion of L-dopa (3,4 dihydroxy L-phenylalanine) to catechol pyruvate and ammonium An assay for the activity of this enzyme involves incubating excess phenol, pyruvate and ammonium with the enzyme sample for a measured length of time. After the termination of the reaction, the L tyrosine present in the sample is quantified by liquid chromatography The following solutions are mixed 500 100 100 100 1.3 MNH4C100M Na+EDTA pH adjusted to 8.5 80 mM Na2SO3 (a reducing agent to stabilize the reaction) 800 mM Pyruvate 1.0mM PLP 500 mM Phenol 100 . At t-0, you add 100 L of an enzyme solution known to have a protein concentration of 8.5 g/mL. After 70 minutes, you add 100 L trichloroacetic acid which terminates the reaction Using the HPLC, you find that the concentration of L-tyrosine in this final solution is 2.3 mM ) What is the activity of the oniginal enzyme solution (in IUmL)? i) What is the specific activity of the oniginal enzyme sobution (in IUmg protein)?Explanation / Answer
Enzyme activity Activity = Units = mol of (Substrate Consumed or Product Formed) / min
Activity concentration (C) = [Activity] = mol/min*ml = Units/ml
mM = µmol/ml
Time = 7 minutes
Activity = (2.3)/7
= 0.328 units/ml
2) Specific Activity = Total Activity/Total Protein = Units/mg
Total Activity = (Activity)(Total Volume) = Units/ml* ml = Units
Therefore, Total Activity = 0.328 units/ml * 100 * 10-3ml = 0.0328 units
Protein weight = 8.5 µg/ml * 100 µl * 10-3 = 0.85 µg = 0.85 * 10-3 mg
Therefore, specific activity = 0.0328 units/ (0.85 * 10-3 mg) = 38.5 units/mg
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