Enzymes Can Be Regulated

activated by AMP

fructose-6-phosphate + ATP

l+ve

i> fructose-1,6-bisphosphate

I -ve inhibited by ATP

Figure 11.9. Phosphofructokinase is regulated by the binding of ATP or AMP at a regulatory site that is separate from the active site. Binding of ATP inhibits while binding of AMP activates.

inactive inactive active kinase 1 inactive inactive inactive active kinase 1 inactive

cyclin B

Figure 11.10. Control of Cdk1 by cyclin B and by phosphorylation.

cyclin B

cyclin B

Figure 11.10. Control of Cdk1 by cyclin B and by phosphorylation.

at threonine 14 and tyrosine 15. In order to become an active enzyme, Cdkl must lose these two phosphates while cyclin B is bound. This complex control means that Cdkl can act as a checkpoint. If the phosphate groups have been removed from T14 and Y15, AND cyclin B is present at a high enough concentration, THEN it is safe to proceed into mitosis.

Cdk1 is an example of an enzyme that is turned off by phosphorylation. Other enzymes are turned on by phosphorylation. Examples include RNA polymerase II (page 120) and glycogen phosphorylase (page 305).

IN DEPTH 11.3 Rapid Reaction Techniques

Enzyme measurements are usually carried out with low concentrations of the enzyme because higher concentrations would give initial velocities too fast to measure. Initial velocity measurements are always made long after the enzymesubstrate complex has formed. It would be very interesting to be able to observe the actual formation of enzyme-substrate complexes and indeed, more generally, to observe the formation of protein-ligand complexes. These reactions are rapid, occurring on a millisecond time scale or less.

Hartridge and Roughton devised the method illustrated in the accompanying diagram for measuring the rate at which hemoglobin combined with oxygen. A ram pushes two syringes, one containing hemoglobin and one containing oxygenated solution. The solutions mix and pass down a tube, so that the further one looks to the right in the tube, the more time the oxygen has had to bind to the hemoglobin. The association of oxygen with hemoglobin can be monitored by an optical change, the same color change that causes arterial blood to be bright red while venous blood is a dark bluish red. Simply moving an optical detector from left to right along the tube showed the concentration of oxygen-hemoglobin complex after different reaction times. However, all the time the measurements are being made, the hemoglobin solution is running out of the pipe at the right-hand end, so that one can only use this approach when large quantities of the protein are available.

The second figure shows a less wasteful method called stopped flow. Here two solutions are mixed and passed into an observation chamber and from it into a syringe. The plunger of this "stopping syringe" moves back until it hits a stopping plate and the flow stops. Observations are made on the solution as it ages in the observation chamber, using a high-speed recording device that is triggered by the stopping syringe as it hits the plate. Stopped flow allows observations of reactions down to about 0.1 ms after mixing. For example, the rate at which calmodulin (page 200) binds calcium can be measured by filling one syringe with calmodulin, the other with a calcium solution. In this case the reaction can be followed because one tyrosine residue on calmodulin (Tyr138) increases its fluorescence when calmodulin binds calcium.

move along pipe to sample reaction at different times after mixing meter move along pipe to sample reaction at different times after mixing meter

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