Homework 1 KEY - Homework Answers#1 1 Assume that the...

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Homework Answers #1 1. Assume that the overall reaction requires two separate catalytic sites, A and B. Suppose further that mutation A - inactivates catalytic site A, and mutation B - inactivates catalytic site B. Then, either of these mutations will prevent the overall reaction if all copies of the protein contain the mutation. Now separate the tetramers of the two mutant proteins into their component monomers, and mix them. The monomers in the mixture have only two possible compositions: A + B - and A - B + , where a superscript “+” denotes the wild-type protein at a particular catalytic site and superscript “-“ again denotes a mutation. The tetramers formed from the mixture can have 5 possible compositions: (A + B - ) 4 , (A + B - ) 3 (A - B + ) 1 , (A + B - ) 2 (A - B + ) 2 , (A + B - ) 1 (A - B + ) 3 and (A - B + ) 4 . If both catalytic steps must occur in the same monomeric subunit, all these hybrid tetramers will be inactive. But if reactions A and B can occur in different in different subunits of the tetramer, the second, third and fourth type of hybrid will be able to catalyze the overall reaction. To evaluate the results quantitatively, you’d have to know the population of hybrids in the reassembled tetramers. If the mutations do not affect the reassembly, the populations should follow a binomial distrution and could be calculated from the concentrations of the two mutant enzymes in the mixture. The analysis could be tested by changing the ratio of the two proteins. 2. The ACP domain must not bind too tightly to any one of the other domains; it has to be able to interact with it but then dissociate and move on to the next domain. Tight binding to one of the sites would interfere with movement to the next site in the cycle. 3. The citrate transporter in the mitochondrial inner membrane moves a dicarboxylic acid such as malate into the mitochondrial matrix in exchange for the citrate that it moves out. The need for this exchange explains the acceleration of citrate efflux by malate (row 5 of the table). Oxaloacetate cannot replace malate (row 4), and does not interfere with the effect of malate (line 6), indicating that it probably does not bind to the transporter. (The mitochondrial inner membrane has no transporter for oxaloacetate.) (The data presented in the table do not exclude the possibility that malate just has an allosteric stimulatory effect on citrate transport. To investigate this point, one could try the experiment in reverse: see whether adding citrate stimulates efflux of malate.
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