bioc441 - 2008AnsProbSet5

bioc441 - 2008AnsProbSet5 - Answers to Problems in Homework...

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Answers to Problems in Homework Set #5 1. Assume that you have expressed pyruvate carboxylase in E. coli and that you can make site-directed mutations in the catalytic site of either the biotin carboxylase (BC) or the carboxyltransferase (CT) domain. Make one strain of bacteria with a mutation that inactivates BC, and another with a mutation that inactivates CT. Either mutation blocks the overall reaction (pyruvate + ATP + CO2 oxaloacetate + ADP). Purify the enzymes from the two mutants, dissociate the tetramers into their monomeric subunits and reassamble tetramers from a mixture of monomers from the two mutants. Consider a hybrid tetramer that has the BC mutation in one of its monomeric subunits and the CT mutation in an adjacent subunit. If the biotin bound to one subunit has access only to the catalytic sites of the same subunit, the reaction would still be blocked. If it has access to BC site of one subunit and the CT site of the adjacent subunit, the hybrid enzyme will be able to catalyze the overall reaction. To interpret 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. 2. The ACP unit must not interact too tightly with any of the catalytic sites. Tight binding to one of the sites would interfere with movement to the next site in the cycle. 3.
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This note was uploaded on 04/15/2010 for the course BIOC 441 taught by Professor Young&parson during the Spring '08 term at University of Washington.

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bioc441 - 2008AnsProbSet5 - Answers to Problems in Homework...

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