The following will be discussed in this chapter: Allosteric regulation, covalent modification, regulation of enzyme activity by subunit association/disassociation, regulation by proteolytic cleavage of proenzyme.
In this chapter, students will be able to: Know the regulatory points in glycolysis (Hexokinase, phosphofructokinase-1, pyruvate kinase), be able to name the allosteric inhibitors and allosteric activators involved in the regulation of each enzyme, understand the relationship between thes allosteric activators/inbitors to the energy state of the cell,...
TheArabidopsis thalianagenome contains four genes encoding NADP-malic enzymes (NADP-ME1–4). Two isoenzymes, NADP-ME2 and
NADP-ME3, which are shown to be located in the cytosol, share a
remarkably high degree of identity (90%). However, they display different
expression patterns and show distinct kinetic properties, especially with
regard to their regulation by effectors, in both the forward (malate oxida-tive decarboxylation) and reverse (pyruvate reductive carboxylation) reac-tions.
A reaction happens need the energy to vibrate the molecules and the reactant concentration enough. The energy here is often provided by heat.
However, in living system, high temperature may harm the biological structure
Truly that the concentration in living system is very low. So living organisms solve these problems by using enzyme
The molecular mechanism of cGMP-dependent protein kinase activation
by its allosteric regulator cyclic-3¢,5¢-guanosine monophosphate (cGMP)
has been intensely studied. However, the structural as well as thermo-dynamic changes upon binding of cGMP to type I cGMP-dependent
protein kinase are not fully understood.
Malonyl-CoA, a potent inhibitor of carnitine pamitoyl
transferase-I (CPT-I), plays a pivotal role in fuel selection in
cardiac muscle. Malonyl-CoA decarboxylase (MCD) cata-lyzes the degradation of malonyl-CoA, removes a potent
allosteric inhibition on CPT-I and thereby increases fatty
acid oxidation in the heart. Although MCDhas several Ser/
Thr phosphorylation sites, whether it is regulated by AMP-activated protein kinase (AMPK) has been controversial.
We therefore overexpressed MCD(Ad.MCD) and consti-tutively active AMPK (Ad.
Fructose 2,6-bisphosphate (Fru-2,6-P2
) plays an important
role in the regulation of major carbohydrate fluxes as both
allosteric activator and inhibitor of target enzymes. To
examine the role of Fru-2,6-P2in the regulation of hepatic
carbohydrate metabolism in vivo,Fru-2,6-P2 levels were
elevated in ADM mice with adenovirus-mediated overex-pression of a double mutant bifunctional enzyme, 6-phos-phofructo-2-kinase/fructose-2,6-bisphosphatase (n¼6), in
comparison to normal control mice (control,n¼6). ...
UDP-galactose 4-epimerase fromKluyveromyces fragilisis a homodimer
containing one catalytic site and one NAD
as cofactor per subunit. One
5¢-UMP, a competitive inhibitor, binds per dimer of epimerase as isolated
and causes inactivation. Addition of 0.2 mminhibitor to the enzyme in vitro
leads to three sequential steps: first, the inhibitor binds to the unoccupied
site; second, the inhibitor bound ex vivo is displaced allosterically; and
finally, both sites are occupied by the inhibitor.
The two main mammalian calpains, 1 and 2, are heterodimers of a large
80 kDa and a small 28 kDa subunit that together bind multiple calcium
ions during enzyme activation. The main contact between the two subunits
of these intracellular cysteine proteases is through a pairing of the fifth
EF-hand of their C-terminal penta-EF-hand (PEF) domains.
Here we report on the role of Glu59 in the fumarate-mediated allosteric
regulation of the human mitochondrial NAD(P)
-dependent malic enzyme
(m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or
Leu. Our kinetic data strongly indicated that the charge properties of this
residue significantly affect the allosteric activation of the enzyme.
Escherichia coli 3-phosphoglycerate dehydrogenase
(PGDH) catalyzes the first step in serine biosynthesis, and is
allosterically inhibitedby serine. Structural studies revealeda
homotetramer in which the quaternary arrangement of
subunits formed an elongated ellipsoid. Each subunit
consisted of three domains: nucleotide, substrate and regu-latory. InPGDH, extensive interactions are formedbetween
nucleotide binding domains.
Cyclic AMP receptor protein (CRP) regulates the expression of more then
100 genes inEscherichia coli. It is known that the allosteric activation of
CRP by cAMP involves a long-distance signal transmission from the N-ter-minal cAMP-binding domain to the C-terminal domain of CRP responsible
for the interactions with specific sequences of DNA. In this report we have
used a CRP mutant containing a single Trp13 located in the N-terminal
domain of the protein.
Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid
pyruvate kinase and thus represents an important regulator of energy meta-bolism in these protozoan parasites. A 6-phosphofructo-2-kinase, respon-sible for the synthesis of this regulator, was highly purified from the
bloodstream form ofTrypanosoma bruceiand kinetically characterized. By
searching trypanosomatid genome databases, four genes encoding proteins
homologous to the mammalian bifunctional enzyme 6-phosphofructo-2-kinase⁄fructose-2,6-bisphosphatase (PFK-2⁄FBPase-2) were found for both
cAMP receptor protein (CRP), allosterically activated by
cAMP, regulates the expression of several genes inEscheri-chia coli. As binding of cAMP leads to undefined conform-ational changes in CRP, we performed a steady-state and
time-resolved fluorescence study to showhow the binding of
the ligand influences the structure and dynamics of the
protein. We used CRP mutants containing a single trypto-phan residue at position 85 or 13, and fluorescently labeled
with 1,5-I-AEDANS attached toCys178.