Question

Question 4 Multi-Part Questions. Make sure to answer ALL parts (a-c)! You have isolated two versions of the same enzyme, a wild type and mutant. The marteses You have determined the following kinetic characteristics for each of the enzymes. Vmax Wild Type Km 200 umol / min 0.2mm Mutant 2 umol / min 20mm and the reaction is a two-step reaction where k.1 is much larger than k3, which enzyme has the higher affinity for substrate to you You determine that the single amino acid change between these enzymes was a glutamic acid residue (found in the wild types b. What are of the enzyme would you expect to find this single amino acid mutation? Explain your reasoning c. Would you expect this enzyme to have an optimal pH for activity? Explain. • T.E.E. • 3 (12pt) TTT Arial

Answer 1

It is known that lesser the value of Km, more is the affinity of the enzyme towards its substrate. Km is the concentration of substrate at which half the maximum velocity is achieved.

Thus the enzyme which has higher affinity for the substrate would have lower Km. Since the wild type enzyme has lower Km than the mutant enzyme, the wild type enzyme has the higher affinity for substrate.

b) Wild type enzyme has glutamic acid which is a polar and charged residue and acidic in nature. The mutant has alanine in place of glutamic acid. Alanine is non polar amino acid. Thus the salt bridge or hydrogen bonding interactions formed by glutamic acid are disrupted when it is replaced by alanine.

c) No, the mutant will not show activity at optimal pH as that of wild type. This is because at optimal pH, the wild type has glutamic acid which has a charged side chain and can interact with other residues via salt bridge interaction or hydrogen bonding. Whereas the mutant with alanine residue would not have any charge to form salt bridge or hydrogen bonding interactions.