Allosteric regulation:
Many enzymes are subject to allosteric regulation; aspartate transcarbamoylase (ATCase) is a particularly good example because it has been studied so thoroughly.
ATCase catalyzes a reaction in the pyrimidine biosynthetic pathway, which supplies cells with UTP and CTP.
ATCase
carbamoyl-P + Asp ------------> carbamoyl aspartate
ATCase has a total of twelve (12) subunits per macromolecular assembly.
These subunits are of two types: a regulatory subunit (R), which has no catalytic activity, and a catalytic subunit, which facilitates the reaction. The two types of subunits can be separated:
p-hydroxymercuribenzoate
R6C6 -------------------------------------> 3
R2 + 2 C3
The X-ray structure of ATCase has recently been completed; it took almost 20 years because of the complexity of the protein. The structure shows that the regulatory site is about 6.0 nm from the active site (a clear example of allostery!).
Regulatory molecules:
ATCase activity is inhibited by CTP; it is activated by ATP. Both of these regulatory molecules bind at the same site on the regulatory subunit.
This regulation achieves two ends:
1. Equalizes the rates of formation of purine and pyrimidine nucleotides
2. Feed -forward activation = activates enzyme when enough ATP is present to synthesize CP.
Covalent modification - bond formation:
Glycogen phosphorylase is an example of an enzyme regulated by its state of covalent modification.
It is found in glycogen granules, which are optically dense bodies in the cytosol containing all of the enzymes for synthesis and degradation of glycogen and some of the control elements.
Glycogen phosphorylase facilitates the breakdown of glycogen by catalyzing a phosphorolysis reaction. In this reaction, phosphate plays a role in breaking the glycosidic bond that is analogous to that played by water in hydrolysis reactions.
glycogen phosphorylase
(glucose)n + Pi <==> glucose 1-phosphate + (glucose)n-1
The glucose-1-phosphate formed here can't be used for glycolysis. Why not?
Sequence of regulatory reactions:
a. epinephrine (adrenaline), produced in the adrenal medulla, circulates in the bloodstream and stimulates the breakdown of muscle glycogen
b. epinephrine binds to a protein receptor in the plasma membrane; the synthesis of c-AMP is stimulated
c. c-AMP binds to a protein kinase, at an allosteric site, to activate this enzyme
d. the protein kinase phosphorylates two enzymes, phosphorylase kinase and glycogen synthetase.
e. phosphorylase kinase is active in the phosphorylated state
f. it converts phosphorylase b (inactive) into phosphorylase a, which is active, by phosphorylating it at amino acid residue 14 (serine).
This pathway amplifies the original signal for glycogen breakdown (the binding of epinephrine to its receptor) and is often called a regulatory cascade.
Inactivation:
The pancreas monitors blood glucose levels, which are normally in the range of 80 - 120 mg/dl. If blood glucose levels rise above this range, insulin is secreted, and glucose enters the liver from the bloodstream.
a. glucose binds to phosphorylase a (the active form)
b. this binding causes an conformation change that exposes the serine 14 -phosphate
c. a phosphatase, which was already bound to the phosphorylase a molecule, is now able to remove the phosphate group as Pi
Covalent Modification - bond cleavage:
Another type of regulation of enzyme activity by covalent modification involves covalent bond cleavage in a zymogen to produce the active form of the enzyme.
An example of this type of regulation is conversion of chymotrypsinogen, which is the zymogen form, into chymotrypsin.
Mechanism of activation:
a. zymogen granules containing chymotrypsinogen are secreted from the pancreas into the lumenal space of the small intestine
b. hydrolysis of the peptide bond of chymotrypsinogen between Arg 15 and Ile 16 exposes a new COO- and HN3+
c. the new amino terminal Ile, which is quite hydrophobic, turns inward and contacts Asp 194.
d. protonation of the new amino terminal NH2 group stabilizes the active form of chymotrypsin.
e. this interaction, in turn, causes Met 192 to move to surface of protein, leaving behind a hydrophobic pocket in which the substrate binds
f. one of the 195-193 pair of NH groups moves to a location where it can stabilize the oxyanion intermediate that forms during the catalytic cycle
Overview:
the duodenum produces enteropeptidase (by contact with food?)
enteropeptidase converts trypsinogen into trypsin
trypsin, in turn, reacts with chymotrypsinogen to form first p-chymotrypsin, and then chymotrypsin
chymotrypsin acts on dietary proteins and peptides to produce amino acids and peptides
Other forms of regulation:
a. regulation of synthesis
b. hormonal regulation of synthesis
c. specific degradation, including PEST sequences and the N-end rules
