Question

30) E. colt' (illustrated in the figure below). Thus, the first enzyme of the pathway will have an active site that binds to the amino acid aonine and an allosteric site that binds to the amino acid s metabolic pathway synthesizing isoleucine from threonine is subject to feedback inhibition Threonine 31) You are interested in harnessing E. coli for the large scale commercial production of the amino acid isoleucine from threonine. You Enzyme A have optimized the growth conditions, but no matter how much threonine you add to the medium, feedback inhibition is limiting the amount of isoleucine produced. What genetic alteration could dramatically increase production of isoleucine ? A) mutate the gene for enzyme A so that the protein fails to fold properly B) mutate the promoter of the operon encoding for enzymes A, B, C, and D so that ơ70 does not bind to it anymore C) mutate the gene for enzyme B so that the enzyme binds more tightly to the substrate (better catalysis) D) mutate the gene for enzyme A so that the enzyme lacks an active site, Enzyme D but is otherwise intact Intermediatel me Intermediate II Intermediate III Isoleucine- mutate the gene for enzyme A so that the enzyme lacks an allosteric site, but is otherwise intact 32) You fail to get the mutant you need, but you discover that intermediate II is chemically stable, cheap to produce, and is readily taken up by the bacterial cells. How does this information provide you with a solution to your yield problems?

Answer 1

30. Enzyme A of the pathway will have an actve site that will bind to the amino acid Threonine and an allosteric site that will bind to the product i.e Isoleucine. End-product inhibition functions to ensure levels of an essential product are always tightly regulated. If product levels build up, the product inhibits the reaction pathway and hence decreases the rate of further product formation.

31.Option E is correct. If the gene for the Enzyme A is mutated such that its allosteric site is deleted but otherwise intact. This would ensure that the allosteric inhibitor i.e Isoleucine would not interfere in the pathway as result there would be uninterrupted increase in product formation.

32. As intermediate II is chemically stable ,cheap to produce and is easily taken up by the bacterial cells. Overproducing this intermediate II would ensure that these intermediate is taken up by the bacterial cells and the process would start from intermediate II thereby eliminating Intermediate I and the need of Enzyme A who have an allosteric site for end product inhibition. Due to lack of enzyme A , the bacterial cells would take up intermediate II and produce Isoleucine and this Isoleucine would not be able to perform end product inhibition.