The Contribution of Each Enzyme to Flux through a Pathway Is Experimentally Measurable

There are several ways to determine experimentally how a change in the activity of one enzyme in a pathway affects metabolite flux through that pathway. Consider the experimental results shown in Figure 15-33. When a sample of rat liver was homogenized to release all soluble enzymes, the extract carried out the gly- colytic conversion of glucose to fructose 1,6-bispho- sphate at a measurable rate. (This experiment, for simplicity, focused on just the first part of the glycolytic pathway.) When increasing amounts of purified hexokinase IV were added to the extract, the rate of gly-colysis progressively increased. The addition of purified PFK-1 to the extract also increased the rate of glycolysis, but not as dramatically as did hexokinase. Purified phosphohexose isomerase was without effect. These results suggest that hexokinase and PFK-1 both contribute to setting the flux through the pathway (hexokinase more than PFK-1), and that phosphohexose isomerase does not.

Similar experiments can be done on intact cells or organisms, using specific inhibitors or activators to change the activity of one enzyme while observing the effect on flux through the pathway. The amount of an

FIGURE 15-33 Dependence of glycolytic flux in a rat liver homog-enate on added enzymes. Purified enzymes in the amounts shown on the x axis were added to an extract of liver carrying out glycolysis in vitro. The increase in flux through the pathway is shown on the y axis.

enzyme can also be altered genetically; bioengineering can produce a cell that makes extra copies of the enzyme under investigation or has a version of the enzyme that is less active than the normal enzyme. Increasing the concentration of an enzyme genetically sometimes has significant effects on flux; sometimes it has no effect.

Three critical parameters, which together describe the responsiveness of a pathway to changes in metabolic circumstances, lie at the center of metabolic control analysis. We turn now to a qualitative description of these parameters and their meaning in the context of a living cell. In Box 15-3 we will provide a more rigorous quantitative discussion.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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