Covalent modification and

allosteric regulation work together.

REGULATORY ENZYMES

    Regulatory enzymes are those which have properties which make them regulators of the rates at which metabolic processes occur.  For instance, phosphofructokinase controls the rate at which the reactions of glycolysis take place.

 

  Two major types of regulatory enzymes:

§      1.  allosteric

§      2.  covalently modulated enzymes

 

1.  ALLOSTERIC ENZYMES

§     The activity of allosteric enzymes is modulated by another molecule called an effector.  The effector binds reversibly and noncovalently to the allosteric enzyme at a site separate from the catalytic site.  Effectors may be positive or negative.  A given allosteric enzyme may have both positive and negative effectors.  Allosteric enzymes are larger, more complex, and difficult to purify.  They are always multimeric.

  

 

Kinetics of allosteric enzymes

n      Allosteric enzymes usually show cooperative binding kinetics.  Most show positive cooperativity.  This type of kinetics is demonstrated in the binding of oxygen to hemoglobin.  Cooperative kinetics begin with a linear, but slow rate of reaction and then accelerate before saturating.  The graph of cooperative kinetics is sigmoidal.

Kinetics of allosteric reactions


2.  COVALENTLY MODULATED REGULATORY ENZYMES

  

   An inactive form is converted to an active form by covalent modifications catalyzed by other enzymes.

   

Examples: 
Covalent activation of zymogens

    Zymogens are large inactive precursors of proteolytic enzymes.  They are synthesized in the pancreas.  If they were not synthesized in an inactive form they would digest the site of their manufacture.  An enzyme exists to activate each zymogen:

                            pepsin

§      pepsinogen                                 pepsin + peptides

                            enterokinase

§      trypsinogen                           trypsin + hexapeptide

                                 trypsin

§      chymotrypsinogen                  chymotrypsin + 2 dipeptides

 

 

 


CONTROL OF GLYCOGEN SYNTHESIS AND BREAKDOWN

   This is an example of metabolic regulation utilizing both allosteric and covalently modified regulatory enzymes.

 

    Glycogen is a highly branched polymer of a-D, glucose linked together by a, 1-4 linkages.  It is present in the muscle and liver of higher animals and serves as a storage form of glucose.  Separate pathways for the synthesis and breakdown of glycogen must be rigorously controlled.  WHY?  One must be turned off when the other is turned on to effect any net change.  

 

Epinephrine Cascade

   Vigorous muscular activity stimulates the release of epinephrine by the adrenal medulla.  The epinephrine will initiate a cascade which will lead to the breakdown of glycogen to glucose in the muscle predominantly and to a lesser extent in the liver.

 

EPINEPHRINE CASCADE

Protein kinase also phosphorylates glycogen synthetase, inactivating it.

REVERSAL

   When glucose is present in a sufficient concentration, the breakdown of glycogen needs to be stopped. 

 

   Glucose binds to phosphorylase a causing it to expose a phosphate which can then be cleaved by another enzyme, phosphorylase phosphatase:

                phosphorylase phosphatase

phosphorylase a                             phosphorylase b

Phosphorylase phosphatase has three substrates.

    Glycogen synthetase is also a substrate for phosphorylase phosphatase.  When phosphorylase a converts to phosphorylase b, phosphatase can not bind to the b inactive form so it is free to bind to glycogen synthetase and activate it by removing a phosphate group.  Phosphorylase phosphatase can also remove a phosphate from phosphorylase kinase, inactivating it.  NOTE:  Phosphorylase phosphatase has three substrates:  1)  phosphorylase a;  2)  glycogen synthetase; and 3)  phosphorylase kinase.

n     

 

 

Phosphorylase phosphatase must be kept inactive to effectively breakdown glycogen to glucose.

   This is accomplished by the following reaction:

 

                   cAMP dependent protein kinase

     inhibitor 1                        inhibitor 1~P, which blocks phosphorylase phosphatase

 

Insulin decreases the amount of inhibitor 1.

   Insulin induces the build up of glycogen in the muscles.  It does so by decreasing the amount of phosphorylated inhibitor 1.  This unleashes the activity of phosphorylase phosphatase which leads to the activation of glycogen synthetase and deactivation of phosphorylase a.

 

 

 

 

cAMP is removed from the membrane.

n      The reversal is made complete by the degradation of the cAMP bound to the cell membrane.  A specific phosphodiesterase converts cAMP to AMP.  Caffeine and theophylline inhibit the activity of phosphodiesterases and thereby prolong the effects of cAMP.

 


AMPLIFICATION

 

   This sequence is an example of amplification.  A small number of epinephrine molecules control the whole process.  If each step had to be regulated by epinephrine directly 1000 X’s more epinephrine would be required.

 

ADDITIONAL CONTROLS

   The controls outlined above are supplemented by two other controls which add flexibility:

1.  Phosphorylase b is active only in the presence of high concentrations of AMP.  AMP is a positive allosteric effector of phosphorylase b.  ATP competes with AMP and thus serves as a negative effector of phosphorylase b.  Glucose-6-phosphate also inhibits phosphorylase be by competing with AMP for the regulatory site.  Therefore, phosphorylase b is usually kept inactive by the presence of ATP and glucose-6-phosphate.

n     

Additional controls:

  2.  Phosphorylase kinase is partially activated by high levels of Ca++ which are present when muscles have been working hard.  One of the subunits of phosphorylase kinase is calmodulin, a calcium binding protein that regulates many enzymes in eukaryotes.