5. Much research has been devoted to understanding the structure and mechanism o

Question

5. Much research has been devoted to understanding the structure and mechanism of action of DNA topoisomerases and their inhibitors. It is known that when they cleave a DNA strand it becomes transiently covalently linked to the enzyme. Shown below is the yeast topoisomerase II active site for DNA cleavage as determined by x-ray crystallography. Arrows and dashed lines indicate the roles of the amino acid residues in DNA strand cleavage. 449 1) Base -1) Base 73 736 781 526 528 782 a. What is the EC number for this enzyme? (Use BRENDA or another of the online protein databases to look it up.) To what enzyme class does it belong? Why? b. Identify each of the 5 amino acid residues involved in the catalytic mechanism that are shown interacting with dashed lines. List their position and three letter abbreviation. Their positions in the enzyme chain are indicated by numbers in the figure. What role(s) do each of the amino acids residues and each of the two metal ions play in the catalytic mechanism? Identify residue 782 and describe its role in the catalytic mechanism A and B are identical and are a metal ion that is among the most common in the body. What metal ion are A and B likely to be. Rationalize your choice. c. d. e. f. What roles do HA and B: play? Could they be water molecules? Explain. g. Draw the structure of the transition state for the phosphate group. What is its geometric name? Was its formation duc to nucleophilic or electrophilic attack on the phosphorus? Explain. h. Draw the structure of the covalent intermediate. Is the linkage to the DNA 3' or 5?

Explanation / Answer

a. EC number= 5.99.1.3. It belongs to isomerase class of enzymes. According to the enzyme commission system of nomenclature, the first digit of EC represents the class to which the enzyme belongs.

b. amino acid residue: 449-glutamic acid (glu), 528- aspartic acid (asp), 526-aspartic acid (asp), 736-histidine (his), 781-arginine (arg).

c. The magnesium ions form salt bridges with both the protein and the DNA. Can also accept other divalent metal cations, such as Mn2+ or Ca2+. Amino acid residues help in DNA binding, transition state stabilizer (781), metal binding (526, 528, 449), catalytic (449).

d. residue 782 is tyrosine (tyr). It is the active site of the enzyme.

e. They are likely to be magnesium ions because they interact with both proteins and DNA.

f. HA is an acid most probably water molecule that extracts the hydrogen from the 3-terminal hydroxyl group. B: has not been identified but it is base that deprotonates the active site.

h. it links to 3' end of the DNA.

Related Questions

Navigate

• Browse (All)
• Browse 5
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.