3 Individual Steps of Metabolic Pathways

Initially the ex­istence of a pathway is identified by observing that the consumption of certain reactants leads to the accumulation of certain products.

For example, the consumption of sugar and the production of carbon di­oxide and alcohol during alcoholic fermentation is a pathway that has been known for centuries; the over­all reaction is,

By quantitative chemical analysis, it is possible to measure the amount of sugar consumed and the amount of CO₂ and ethanol produced.

Though such an analysis reveals that all the carbon in the sugar may be accounted for in these two products (i.e., no other carbon-containing products are formed), it does not tell us anything about the number or kinds of individ­ual steps that may characterize the overall process.

Also not revealed are coupled reactions such as the formation of ATP during fermentation. The individual steps of a pathway may be revealed using marker and tracer techniques and through the isolation and iden­tification of specific enzymes associated with the pathway.

Marker and Tracer Techniques:

A frequently used technique for identifying the steps in a pathway is to follow the metabolism of a substrate molecule in which one of the atomic positions has been “labeled” with a radioactive isotope or in rare instances a “heavy” isotope (such as deuterium, ²H, or heavy nitrogen, ¹⁵N).

Such a labeled substrate is said to be a tracer because specialized techniques are available to identify these labeled compounds. The tracer is then made available to the cell and metabo­lism allowed to proceed. After a short period of time, metabolism is halted by either rapidly dropping the temperature of the cells or by adding metabolic inhibi­tors.

The cells are then broken open and potential chemical intermediates of the metabolic pathway are isolated. This procedure usually involves centrifugal fractionation of the cells followed by some combination of extraction and chromatographic proce­dures The intermediates peculiar to the pathway being studied can be distinguished from other chemical substances present on the basis of their isotope content (e.g., by their radioactivity) and can then be chemically identified.

Because in a pathway such as substrate*→B* →*C*→D* →E*→product* (10-2) the reaction forming B occurs before the reaction forming C, which in turn occurs before the reaction forming D (and so forth), the amount of time that elapses between the addition of the tracer to the cells and the time at which metabolism is halted can be used to reveal the correct sequence.

In this type of tracer experiment, the labeled substrate is made avail­able at a designated time, and the reactions are al­lowed to proceed for only a short interval so that the labeled substrate can participate in no more than the first one or two reactions of the pathway.

As a result only a few of the intermediates formed will contain the label. Isolation and analysis of the labeled inter­mediates are then carried out. The experiment is re­peated using another sample of cells and labeled sub­strate but is allowed to proceed for a longer period of time before being halted.

As a result an additional (la­beled) intermediate may be identified. The experi­ment is repeated again and again, each time revealing new intermediates of the pathway. When it is suspected that after one or two reactions a substrate may be split into two or more different products,

then the atoms at various positions in the substrate may be labeled so that the products entering each branch may be identified and followed. In the above di­agram, the labeled positions are represented by the symbols * and +. Because some intermediates are not formed in sufficient amounts to permit efficient ex­traction and identification, non-labeled intermediates may be added to the fractionated cells. The non-labeled intermediate mixes with the labeled intermediate and thereby provides sufficient “carrier” for extraction and analysis; this technique is known as isotopic trap­ping.

Another method used to verify the position of an in­termediate in a pathway is by overloading. For exam­ple, suppose that the position of intermediate C in re­action sequence 10-2 is uncertain. To clarify the role of C, two parallel experiments are performed. In one experiment, the substrate is labeled and the amount of label appearing in the product is then followed as de­scribed earlier. In the second experiment, a large amount of intermediate C is added to the cells along with the labeled substrate.

If C is indeed an intermedi­ate in the pathway, then the amount of label recovered in products formed beyond the step that produces C will be greatly diminished. This is because quantities of both labeled and unlabeled C will be incorporated into the subsequent intermediates.

Enzyme Techniques:

Because nearly all metabolic reactions in cells are cat­alyzed by enzymes, it should be possible to identify an enzyme for each metabolic reaction. In enzyme stud­ies of this type, cells are disrupted and a cell-free homogenate prepared. After adding selected substrates and cofactors to the homogenate, the presence of a particular enzyme can be demonstrated by measuring the rate of disappearance of the substrate of that en­zyme, or the rate of appearance of a specific product.

The homogenate can be fractionated by centrifugation into its components, thereby isolating the mito­chondria, plasma membranes, ribosomes, other or­ganelles, or the cytosol. Each of these fractions can be tested for enzymatic activity so that the native loca­tion of the reactions in intact cells may be established.

Use of Enzyme Inhibitors and Enzyme-Deficient Cells:

Yet another approach to determining metabolic reac­tion sequences is through the use of specific enzyme inhibitors or mutant cells that fail to produce a spe­cific enzyme. Numerous enzyme inhibitors are known that block specific reactions steps. In the conversion of substrate A to product E, that is,

an inhibitor that blocks the enzyme catalyzing reac­tion 2 would also prevent the formation of final prod­uct E.

However, even in the presence of an inhibitor one should still be able to make the following two ob­servations:

(1) Intermediate B should continue to be formed from A, because step 1 is not inhibited; in fact, B may even be found to accumulate in excess; and

(2) Addition of an exogenous source of C or D should be accompanied by the continued production of final product E. The use of an inhibitor for one of the reac­tions of a metabolic pathway thus provides a means for identifying the intermediate before the block.

In­hibitors can also be used to test for suspected inter­mediates that come after the block. In a similar manner, a mutant cell that lacks the ge­netic information for producing a specific enzyme may serve as a test organism for studying the steps of the metabolic pathway involving that enzyme.