Question

Christian Anfinsen investigated the structure of bovine pancreatic ribonuclease A in 1950s, and discovered that the amino acid sequence of a protein determines its native conformation and the conformation determines its biological activity. In his experiment, he denatured bovine pancreatic ribonuclease A in an aqueous solution containing the denaturant urea, and the reducing reagent b-mercaptoethanol. The denatured enzyme completely lost its enzymatic activity. On exposure to air after removal of the denaturant and b-mercaptoethanol, the ribonuclease spontaneously reoxidised and regained its full enzymatic activity.

Answer the questions below:

a) Describe the structures of bovine pancreatic ribonuclease A (PBD ID: 1FS3). In your answer you will need to have accessed the Protein Databank and the related research paper.

b) How does urea and b-mercaptoethanol denature the enzyme?

c) What types of amino acids would be expected in the active site of bovine pancreatic ribonuclease A? (Justify your answer based on the chemistry of the substrate-enzyme interactions).

Answer 1

A.

Classification. : HYDROLASE

Organism(s) : Bos taurus (Feral cattle)

Expression System : Escherichia coli

Sequence length : 124

B. Treatment of RNase with mercaptoethanol reduces RNAse's disulfides to sulfhydryls. Subsequent treatment of RNase with urea disrupts hydrogen bonds and allows the protein to be denatured.

C. In the active site, the side chains of His12, His119, Lys41, Asp121, and Gln11, and the main chain of Phe120 are proximal to the vanadyl
group. The apparent roles of these six active-site residues in catalysis.