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Solving Enzyme Structure Problems

Solving Enzyme Structure Problems

The structure of the enzyme Tryptophan Synthetase has been studied extensively by a variety of methods.

In a series of studies, Yanofsky and co-workers examined the effect on enzyme activity of various amino acid changes in the protein sequence (Federation Proceedings, 22:75 (1963) and Science 146:1593 (1964)). Altered amino acids are shown in bold. "Wild-type" is the normal strain isolated from the wild.

Here are two possible explanations for these results:

i) the gly -> glu and gly -> arg changes introduce a charge (+) or (-) into a region of the protein that requires an uncharged amino acid like glycine.

ii) the gly -> glu and gly -> arg changes introduce much larger amino acid side-chains into a space in the protein that requires a small amino acid like glycine.

Yanofsky & co. collected more mutants and examined their proteins to determine which of the above explanations was more likely to be correct:

a)Which of their models is supported by this data? Why?

This is an example of real experimental data, which is often ambiguous, unclear, and difficult to interpret.

Here, you must decide between two models for why these amino acid changes result in an inactive enzyme: the new amino acid side chain is charged instead of neutral; or the new amino acid's side chain is larger than glycine's.

The first data set is ambiguous, because the side chains of the added amino acids are both big and charged. However, the second data set shows that substituting valine, whose side chain is larger than glycine but uncharged, results in an inactive enzyme. This indicates that it is the size of the amino acid side chain at position A that matters, not it's charge (model ii).

One possible explanation for this is that position A is inside the molecule, in a small pocket. This pocket is so small that only small side chains like those of glycine can fit there. Substituting an amino acid with a larger side chain overfills the pocket, changing the conformation of the molecule so that it is no longer able to function.

b) Alterations of amino acids at another location in the protein were found to interact with alterations at position A.

Explain the behavior of mutant 6 in terms of your model of part b.

Mutant 1 is inactive because the glu side chain is too big to fit in the pocket where gly normally sits. Changing the amino acid at position B partially reverses this effect. A possible explanation for this is that the amino acids at positions A and B interact somehow, so that changes in one can compensate for changes in the other.

The simplest model for this is that the side chains of both A and B sit in the same pocket. Therefore, changing gly --> glu (M1) overfills the pocket, resulting in inactive enzyme. The effects of this can be partially reversed by changing tyr --> cys, which opens up more space in the pocket to accommodate the side chain of glu.

c) Given your above model, explain the lack of activity found in mutant 7.

In this case, it appears that reducing the size of a side chain in the pocket (tyr --> cys) also results in formation of inactive enzyme. Therefore, the pocket has to be a certain size for the enzyme to function: gly --> val overfills it and tyr --> cys underfills it. Given the compact nature of protein structures, these results are complex but not surprising.

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