Analysis of Single-Step Pathways

The investigation of a single-step pathway usually begins with a crude cell-free extract from a source abundant in the enzyme that catalyzes the conversion. Two of the most popular sources of cells for many biochemical studies are rat liver and E. coli. To investigate a particular reaction, the precursor (substrate) is added to the extract, and the amount of product formed as a function of time is determined. Once an assay for disappearance of precursor and appearance of product has been developed, the crude extract can be processed into fractions that can be tested to see which are active in the conversion. Through further fractionation and assays it should ultimately be possible to purify the enzyme of interest. Some procedures followed in enzyme purification were discussed in chapter 6, and many procedures used to determine the mechanisms of action of the purified enzymes were considered in chapters 8 and 9. 

The process of assaying for a particular reaction during purification may be complicated in cases requiring cosubstrates, coenzymes, or cofactors. Usually these additional requirements are discovered by the trial-and-error procedure of adding test substances to the reaction mixture and observing whether they accelerate the reaction or lead 

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Analysis of Single-Step Pathways Analysis of Multistep Pathways... 

Different procedures are used for analysis of simple and complex pathways. Analysis of single-step pathways often begins with the isolation and characterization of the enzyme involved. During enzyme isolation each purification step is monitored by a specific assay that measures the conversion of substrate to product. Multistep pathway analysis ideally begins with complementation analysis, a genetic technique that entails the isolation of mutants with genetic blocks in each step of the pathways. Once the numbers of enzymes and intermediates are established, each enzyme can be isolated with the help of a specific assay. 

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