Analysis of Multistep Pathways

The next phase in pathway analysis involves biochemical procedures. As many metabolic intermediates as possible are isolated, their structures are determined, the order of reactions is determined, and the enzymes that catalyze the different reaction steps are isolated and characterized. The mutants isolated for the complementation analysis are also [Pg.237]

The tryptophan biosynthetic pathway in E. coli. There are five enzymatically catalyzed reactions involved in tryptophan biosynthesis and five different polypeptides associated with these reactions. Polypeptides E and D normally make a tetrameric complex, which [Pg.238]

From this point on there are numerous ways of proceeding with the analysis. Crude extracts can be made from different mutant cells. The extract from one mutant can be used to complement the extract from another mutant in tryptophan synthesis, which can lead to an assay for a particular enzyme carried by one mutant that is missing in the other mutant. The goal at this juncture is to purify each enzyme of the pathway so that the properties of the enzymes and the reactions they catalyze can be individually scrutinized. All of the tryptophan enzymes have been isolated from E. coli. Studies of other systems indicate a remarkable similarity for the operation of this pathway in different microorganisms and plants. [Pg.238]

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