Download Chapter 7 7 The Behavior of Proteins: Enzymes Mechanisms and

Mary K. Campbell
Shawn O. Farrell
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Chapter 7
The Behavior of Proteins:
Enzymes Mechanisms
Enzymes,
Mechanisms, and Control
Paul D. Adams • University of Arkansas
The catalytic activities of many enzymes
are regulated by
1. Feedback inhibition (products – reactants)
2 Allosteric
2.
All t i regulation
l ti (allosteric
( ll t i iinhibitor
hibit and
d
allosteric activator)
3. Covalent modification (phosphorylation)
4. Proteolysis (eg. Zymogen)
5. Coenzyme
1. Feedback Inhibition
Formation of product
inhibits its continued
production
Aspartate transcarbamoylase
(ATCase) feedback inhibition
product
2. Allosteric regulation
• Allosteric: Greek allo + steric, other shape
• Allosteric enzyme:
enzyme: an oligomer whose biological activity is
affected by other substances binding to it
ese subs
substances
a ces change
c a ge the
ee
enzyme’s
y e s ac
activity
y by a
altering
e g
• these
the conformation(s) of its 4°structure
• Allosteric effector:
effector: a substance that modifies the behavior of
an allosteric enzyme; may be an
• allosteric inhibitor
• allosteric activator
Allosteric Enzymes
• The key to allosteric behavior is the existence of multiple
forms for the 4°structure of the enzyme
y
• allosteric effector (allosteric inhibitor or allosteric
activator):
): a substance that modifies the 4° structure
activator)
of an allosteric enzyme
• homo
homotropic
o ot
ottropic
op c effects:
e ects tthe
e subst
substrate
ate a
also
so se
serves
es as a
positive (stimulatory) modulator, or activator; e.g., the
binding
g of aspartate
p
((its substrate)) to ATCase. ((tends
to be positive regulator)
• hetero
heterotropic
tropic
p effects: allosteric interactions that occur
when different substances are bound to the protein;
e.g., inhibition of ATCase by CTP and activation by
ATP
Allosteric Enzymes
„Allosteric
enzymes do not obey Michaelis-Menten kinetics.
• Two types of allosteric enzyme systems exist
Note: for an allosteric enzyme, the substrate concentration
at one-half Vmax is called the K0.5 ((與Km有所區隔）
• K system:
system
y
: an enzyme
y
for which an inhibitor or activators
alters K0.5
• V system:
system: an enzyme for which an inhibitor or activator
alters Vmax but not K0.5
Substrate-activity
y curves for representative
p
allosteric
enzymes. (a) The sigmoid curve of a homotropic enzyme, in
which the substrate also serves as a positive (stimulatory) modulator,
or activator.
i
Note
N the
h resemblance
bl
to the
h oxygen-saturation
i curve off
hemoglobin.
K system
positive modulator
( ll t i activator)
(allosteric
ti t )
negative modulator
(allosteric inhibitor)
The effects of a positive modulator (+) and a negative modulator (–) on an
allosteric enzyme in which K0.5
0 is altered without a change in Vmax. The
central curve shows the substrateactivity relationship without a modulator.
V system
Substrate-activity curves for representative allosteric
enzymes. (c) A less common type of modulation, in which Vmax is
altered and K0.5 is nearly constant.
ATCase (Aspartate transcarbamoylase)
• Rate of ATCase catalysis vs substrate concentration
• Sigmoidal shape of curve describes allosteric behavior
• CTP (allosteric inhibitor); ATP (allosteric activator)
ATCase
• Organization of ATCase
• catalytic unit: 6 subunits organized into 2 trimers
• regulatory unit: 6 subunits organized into 3 dimers
Two Models Suggest Mechanisms for
Cooperative Binding
The 1st model: MWC model (concerted model) .
The 2nd model, the sequential model .
MWC model
T form
All subunits switch
from TÆR in unison
Sequential Model
R form
TÆR Transition only occur in
subuints containing bond ligand
The Concerted Model (MWC model)
• Wyman, Monod, and Changeux - 1965
• The enzyme has two conformations
• R (relaxed): the active form; binds substrate tightly
• T (tight
(ti ht or taut):
t t) the
th inactive
i
ti form;
f
binds
bi d substrate
b t t
less tightly
• in
i th
the absence
b
off substrate,
b t t mostt enzyme molecules
l
l
are in the T (inactive) form
• the
h presence off substrate
b
shifts
hif the
h equilibrium
ilib i
ffrom
the T (inactive) form to the R (active) form
• in changing from T to R and vice versa, all subunits
change conformation simultaneously; all changes are
concerted
t d （一致）
（ 致）
Sequential Model (Cont’d)
• Main Feature of Model:
• the binding of substrate induces a conformational
change from the T form to the R form
• the change in conformation is induced by the fit of the
substrate to the enzyme,
enzyme as per the induced-fit
induced fit model
of substrate binding
• sequential model represents cooperativity
3. Reversible Covalent modification—
Phosphorylation
• The side chain -OH groups of Ser, Thr, and Tyr can form
phosphate
p
p
esters
• Phosphorylation by ATP can convert an inactive precursor into
an active enzyme
protein kinases
protein phosphatases
Example: Membrane Transport
Source of PO4 is ATP
• When ATP is hydrolyzed, energy released that allows other
energetically unfavorable reactions to take place
• PO4 is donated to residue in protein by protein kinases
4. Proteolysis-- Zymogen
• For some enzymes, an inactive precursor called a
zymogen is cleaved to form the active enzyme.
• Chymotrypsinogen (zymogen, inactive enzyme)
• synthesized and stored in the pancreas
• a single polypeptide chain of 245 amino acid residues
cross linked by five disulfide (-S-S-)
( S S ) bonds
• when secreted into the small intestine, the digestive
y
trypsin
yp
cleaves a 15 unit p
polypeptide
yp p
from the Nenzyme
terminal end to give π-chymotrypsin
Activation of chymotrypsin
• Activation of chymotrypsinogen by proteolysis
Chymotrypsin
-- a serine protease
-- Because Ser-195 and His-57 are required for activity,
th mustt b
they
be close
l
tto each
h other
th iin th
the active
ti site
it
-- Catalytic triad: His 57, Asp 102, Ser195
-- catalyzes the carboxyl side of aromatic side chains
(Tyr, Phe, Trp)
Chymotrypsin (Cont’d)
•
The active site of chymotrypsin
shows proximity of 2 reactive a.a.
Active Sites and Transition States
•
Enzyme catalysis
• an enzyme provides an alternative pathway with a lower
activation
i i energy
• the transition state often has a different shape than either the
substrate(s) or the product(s)
• “True nature” of transition state is a chemical species that is
intermediate in structure between the substrate and the product.
•
•
T
Transition
iti state
t t analog:
l
a substance
b t
whose
h
shape
h
mimics
i i th
thatt off a
transition state
In 1969 Jenks proposed that
• an immunogen would elicit an antibody with catalytic activity if
the immunogen mimicked the transition state of the reaction
• the first catalytic antibody or abzyme was created in 1986 by
Lerner and Schultz
*(Biochemical Connections, p. 196)
Induced fit model
Enzyme Specificity
• Absolute specificity
specificity: catalyzes the reaction of one
unique substrate to a particular product
• Relative
R l i specificity:
specificity
ifi i : catalyzes
l
the
h reaction
i off
structurally related substrates to give structurally
related
l d products
d
• Stereospecificity: catalyzes a reaction in which one
stereoisomer is reacted or formed in p
preference to all
others that might be reacted or formed
Asymmetric binding
• Enzymes can be
stereospecific
p
((Specificity
p
y
where optical activity may
pay a role)
• Binding sites on enzymes
must be asymmetric
5. Coenzymes
• Coenzyme: a nonprotein substance that takes part in an
enzymatic
y
reaction and is regenerated
g
for further reaction
• metal ions- can behave as coordination compounds. (Zn2+,
Fe2+)
• organic compounds, many of which are vitamins or are
metabolically related to vitamins (Table 7.1).
NAD+/NADH
• Nicotinamide adenine
dinucleotide ((NAD+) is used
in many redox reactions in
biology.
• Contains:
1) nicotinamide ring
2) Adenine ring
3) 2 sugar-phosphate groups
NAD+/NADH (Cont’d)
• NAD+ is a two-electron oxidizing agent, and is
reduced to NADH
• Ni
Nicotinamide
i
id ring
i iis where
h
reduction-oxidation
d i
id i
occurs
• The B6 vitamins are coenzymes involved in amino group
transfer from one molecule to another.
• Important in amino acid biosynthesis
Some important nouns
§ Proenzyme (Zymogen ) : the inactive or nearly
inactive precursor of an enzyme, converted into an
active
ti enzyme b
by proteolysis.
t l i
§ Holoenzyme: An active, complex enzyme
consiting of an apoenzyme and a coenzyme
Holoenzyme = apoenzyme +coenzyme (or cofactor)
§ Coenzyme: low molecular weight organic
molecules (NAD+, FAD+…)
§ Cofactor: metal ions,
ions is required for holoenzyme’s
holoenzyme s
activity (metalloenzyme)
§ Prosthetic g
group:
p the nonprotein
p
component
p
of a
conjugated protein, as the heme group in
hemoglobin
§A ti ti factor:
§Activation
f t
only
l affect
ff t the
th rate
t off reaction
ti