Pathway determination by biochemical genetics

The use of genetic mutants determined the complex pathways that lead to the amino acids. A mutant is an organism that has a different DNA sequence from its parent(s). Mutant bacteria that require a specific compound for growth are called auxotrophs. The first step in pathway determination is to assemble a large collection of auxotrophic mutants that can t make the compound of interest. 

The same logic was used to identify the groups of mutants who allowed the growth of other mutants, or who were able to grow on the compounds excreted by other groups of mutants. In the case of tryptophan, five such complementation groups existed. 

From these data, it should be fairly easy to deduce that complementation group E comes last in the pathway while complementation group A comes first. 

This did not establish the pathway completely. The next step was to identify the compounds that are excreted and allow growth of the other mutants. Next, the pathway was established biochemically by identifying and purifying the individual proteins that carry out the steps corresponding to each complementation group, showing that the enzymes behaved kinetically in the way that the other analyses predicted was necessary. These studies showed that the proteins 

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