Question

33. Enzyme X contains 5 cysteines (A, B, C, D, E). Each cysteine interacts with a specific partner - either a different cysteine or the enzyme substrate. Treatment with beta- mercaptoethanol destroys enzyme functionality. You create five mutant variants in this enzyme; in each, one of the cysteines has been replaced with an alanine. You experimentally characterize each mutant, comparing it to the wild-type enzyme. Change in temperature Enzyme needed to denature KMMUT - KMWT variant protein (TMMUT-TMWT) | CysA+Ala -5°C CysB>Ala -10°C CysCAla 0 CysD=Ala O CysE+Ala For each cysteine, predict whether it interacts with a different cysteine (identify the interacting cysteine) or the substrate. Explain why you made these predictions in one sentence per cysteine or cysteine pair. Do you predict that enzyme X is cytoplasmic or extracellular? Explain in one sentence. (5pts) o +++ -5°C -10°C

Answer 1

33.- To answer this we need to be clear on the function of this amino acid. Cysteine has a thiol lateral group, this thiol has the ability to react with another cysteine's thiol forming a disulfide bridge, which is a very important in protein structure. If a cysteine is participating in a disulfide bond and we replace it with alanine then our protein's structure will be much more weak, leading a being denatured more easely (we need less temperature to denature the protein). If the

Cys A. This one interacts with another cysteine, actually Cys D. We know that because if we replace either A or D we will find the same structural change that leads to the same temperature change in denaturing.

Cys B. This one interacts with another cysteine, actually Cys E. We know that because if we replace either B or E we will find the same structural change that leads to the same temperature change in denaturing.

Cys C. This one interacts with the substrate, its replacement didn't affect the denature temperature.

Cys D. This one interacts with another cysteine, actually Cys A. We know that because if we replace either A or D we will find the same structural change that leads to the same temperature change in denaturing.

Cys E. This one interacts with another cysteine, actually Cys B. We know that because if we replace either B or E we will find the same structural change that leads to the same temperature change in denaturing.

Enzyme X is extracellular, as it is typical that disulfide bonds occur in such extracellular proteins.

34.- Okay, P50 is the oxygen pressure needed to have hemoglobin 50% saturated. Adult hemoglobin needs 25 torr for that, and fetal hemoglobin only needs 18 torr. That means feltal hemoglobin is 1.38 times more saturated at 25 torr oxygen levels.