Chapter 7 Enzyme Regulation BIOCHEM

pyrimidines and purines

aromatic groups that compose
the unique bases found in DNA and RNA

aspartate transcarbamoylase (ATCase)

catalyzes the
first step in pyrimidine biosynthesis

committed step

step in the pathway where the
products of the reaction are committed to the ultimate
synthesis of end products in the pathway
– irreversible under cellular conditions
– catalyzed by allosteric enzymes

feedback inhibition

inhibition of an enzyme
by the end product of
the pathway
– example: ATCase
inhibition by CTP
• ensures pathway
intermediates are not
needlessly formed

allosteric (or regulatory) sites

sites distinct from the
active site at which substrate bind

sigmoidal curve

ex. CTP to ATCase

subunits of ATPcase

6 catalytic and 6 regulatory —> 2 trimers + 3 dimers c6r6

p-hydroxymercuribenzoate

dissociates the enzyme
into:
– a catalytic subunit
consisting of three chains
(c3).
– a regulatory (r) subunit of
two chains (r2).

at the cysteine residue

PALA
N-(phosphonacetyl)-L-aspartate

potent competitive inhibitor of ATCase
– binds to and blocks the active sites
– binds at the boundaries between pairs of c chains within a catalytic trimer
• Each catalytic trimer contributes 3 active sites to the enzyme.

structural
changes that
convert the
compact, less
active tense (T)
state into the
expanded, more
active relaxed
(R) state.

allosteric coefficient (L)

L= T/R ratio of the concentration of
enzyme in the T state to that in the R state

T state

– has a low affinity for substrate.
– has low catalytic activity.
– is favored in the absence of substrate.

R state

has higher affinity for substrate.
– is the most active form.
– is favored upon binding of substrate.

cooperativity

property by which the subunits cooperate
with one another

homotropic effects

cooperative effects of substrates on
allosteric enzymes
– act on enzyme active sites
– generate the sigmoidal curve
– impact ATCase activity by altering the T/R ratio

threshold effect

the
activity of allosteric
enzymes (red line) is
more sensitive to
changes in [S] near KM
than are Michaelis–
Menten enzymes (blue
line) with the same
Vmax

Binding of CTP to the regulatory site of ATCase

favors the T state.
– decreases net enzyme activity.
– increases L from 200 to 1250.

-increases initial phase of sigmoidal curve

ATP allosteric activator of ATcase

binds to the same
site as CTP
– favors the R state
– increases net
enzyme activity

heterotrophic effects

effects of nonsubstrate molecules
on allosteric enzymes

bind at sites other than the active site
– shift the KM
– impact ATCase activity by altering the T/R ratio

isozymes (isoenzymes)

enzymes that differ in amino
acid sequence that catalyze the same reaction
– typically display different kinetic parameters or respond to
different regulatory molecules
– encoded by different genes
– may be expressed in a tissue-specific or development
stage-specific pattern

lactate dehydrogenase (LDH)

tetrameric protein that
catalyzes a step in anaerobic glucose metabolism and
glucose synthesis
• humans have two isozymic polypeptide chains for LDH:
– H protein is highly expressed in heart muscle.
– M protein is highly expressed in skeletal muscle.

covalent modification

covalent attachment of a
modifying functional
group to an enzyme
– most are reversible
– examples:
phosphorylation,
dephosphorylation,
acetylation,
deacetylation

protein kinase

catalyze the phosphorylation of protein substrates by attaching a phosphoryl group to Ser,Thr, Tyr usually ATP

consensus sequence

Individual kinases may recognize many related amino
acid sequences
– Exact sequence is not required (X and Z can vary).
• example: protein kinase A recognizes Arg-Arg-X-Ser-Z or
Arg-Arg-X-Thr-Z
– X = a small residue
– Z = a large hydrophobic residue
– Ser or Thr = the site of phosphorylation

protein phosphatases

catalyze the removal of
phosphoryl groups attached to proteins by hydrolyzing
the bond attaching the phosphoryl group

Phosphorylation is highly effective

the free energy of phosphorylation is large.
– a phosphoryl group adds two negative charges to a protein,
potentially altering electrostatic interactions.
– a phosphoryl group can form 3+ hydrogen bonds.
– kinetics of phosphorylation and dephosphorylation can be
adjusted to meet timing needs of a particular process.
– phosphorylation can be used to amplify signals.
– ATP is the cellular energy currency.

Epinephrine (adrenaline)

induces the "fight-or-flight"
response in muscles.
– Epinephrine triggers the
formation of the intracellular
messenger cyclic AMP
(cAMP)

cAMP

cAMP activates protein
kinase A (PKA).
– PKA phosphorylates target
proteins to alter activity. 4 structure

PKA

phosphorylates target protein, R2C2

pseudosubstrate sequence

sequence that closely
matches the consensus sequence

Cushing's syndrome

a collection of diseases resulting
from excess cortisol secretion by the adrenal cortex
– characterized symptoms such as muscle weakness,
thinning skin that is easily bruised, and osteoporosis
• commonly caused by mutations within the C subunit
disrupt the interaction between the R and C subunits
– results in PKA being active in the absence of cAMP

zymogen (proenzyme)

inactive precursor of proteolytic cleavage

enteropeptidase

Trypsinogen is converted to active trypsin, Trypsin activates more trypsinogen and other zymogens

serpins

(serine protease
inhibitors) = a specific
type of protease inhibitor

α1-Antitrypsin

protects tissues from digestion by
elastase.

emphysema

deficiency in α1-Antitrypsin, leads to excess elastase, damage of alveolar wall by digesting connective tissue proteins

cigarette smoking effects

smoke oxidizes Met 358 of inhibitor of methionine sulfoxide, essential for binding elastase, so double whammy since double the extra elastase

enzymatic cascade

a series of zymogen activations
– amplifies the signal at each step
– used to achieve a rapid response

hemostasis

the process of blood clot formation and
dissolution

intrinsic pathway

activated by exposure of anionic
surfaces upon rupture of the endothelial lining of the
blood vessels

extrinsic pathway

initiated when trauma exposes
tissue factor (TF), an integral membrane glycoprotein
– appears to be most crucial in blood clotting

Factor X

converts the zymogen prothrombin into
thrombin (positive feedback)

Prothrombin 4 domains

gla domain = rich in Ca2+-binding γ-carboxyglutamate (gla)
residues
– 2 kringle domains = help maintain prothrombin in the
inactive form
– serine protease domain

Ca2+ role

binding to Gla brings zymogen prothrombin close to factor X and catalyze conversion into thrombin

fibrinogen

large blood plasma glycoprotein composed of 3 globular regions connected by 2 rods

thrombin role

cleaves
fibrinogen, yielding
A and B peptides
(fibrinopeptides)
and a fibrin
monomer ((αβγ)2)

Fibrin

monomers polymerize, forming a "soft clot."
– stabilized by cross-linking of Lys and Gln residues by
transglutaminase (factor XIIIa)

Vitamin K

γ-Glutamyl carboxylase requires vitamin K to convert Glu
residues in prothrombin to γ-carboxyglutamate.
• γ-Carboxyglutamate is required for prothrombin
activation.

anticoagulant drugs

medicines that reduce
blood clot formation

warfarin

(vitamin
K antagonist) inhibits
enzymes required to
regenerate the active form
of vitamin K

heparin

increases the rate of formation
of irreversible complexes between antithrombin III and
serine protease clotting factors.