The figure shows the catalytic intermediates in the reaction

The figure shows the catalytic intermediates in the reaction of a novel serine carboxypeptidase adapted for degradation of an analogue of the antibiotic microcin C7

Show all steps to the solutions of the following problems.

a. What are the amino acid residues at positions 118, 220, 243, 247, 294 and 311? Provide their full names, three-letter and single-letter abbreviations.

b. The 4 panels (1-4) represent steps in the proposed reaction mechanism. They have been scrambled. In what order should they be arranged? What is the role of Ser-188 and His-311 in the catalytic cycle?

c. In which EC class the enzyme belong?

d. The catalytic activity towards a substrate analogue (DSA) is studied and the following values obtained

ND=no activity

What is the effect of the mutations on the efficiency of the enzyme compared to the wild-type? What information can you obtain?

e. What is the rationale for mutating the residues present in the wild-type protein to alanine?

Gly-22 .118 HN Gly-21 NH2 NH HN-P-O NH2 NH, Ho OH NH NH3 o Gly-22 HN Gly-21 118 NH2 \"H2N o NH NH2 Ho oH NH NH3 .247 311 118 243 *H2N HN-P- NH NH Ho OH NH3 220 NH,\" microcin C7 analogue 311 243 118 NH NH O NH2 H2N o Ho NH2 NH3 NH HO OH 220 NHs NH3 247

Solution

a) The amino acids residue at different positions are given as-:

i) 118-Serine, Ser

ii) 220-Glutamine, Gln

iii) 243-Carboxylic acid

iv) 247-Lysine, lys

v) 294-Arginine,Arg

vi)311-Histidine,His

b) The order of the proposed reaction mechanism should be

b-a-d-c

The role of Ser -118 is it acts as enzyme and His-311 acts as substrate for the enzyme in the caralytic cycle.

c) EC class for the enzyme Serine Carboxy peptidase is EC number 3.4.16.

d)The effect of the mutations on the efficiency of the enzyme is that the catalytic efficiency of enzyme decreased as compared to wild type.

The catalytic efficiency is given by kcat/km

For wild type catalytic efficiency =6.96/74.31=0.09

After mutation the catalytic efficiency decreaed to 0.01,0.02 and 0.

Also there is increase in km value of enzyme as compared to wild type which indicates the enzyme substrate binding capacity decreased after mutation.