Transcript Document

Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Seven
The Behavior of Proteins:
Enzymes, Mechanisms, and Control
Paul D. Adams • University of Arkansas
Allosteric Enzymes
• Allosteric: Greek allo + steric, other shape
• Allosteric enzyme: an oligomer whose biological activity is affected by
other substances binding to it
• these substances change the enzyme’s activity by altering the
conformation(s) of its 4°structure
• Allosteric effector: a substance that modifies the behavior of an allosteric
enzyme; may be an
• allosteric inhibitor
• allosteric activator
• Aspartate transcarbamoylase (ATCase)
• feedback inhibition
_____________ ________________
____________________
____________________
____________________
____________________
____________________
____________________
ATCase
• Rate of ATCase catalysis vs
substrate concentration
• Sigmoidal shape describes
allosteric behavior
• ATCase catalysis in presence
of CTP, ATP
ATCase (Cont’d)
• Organization of ATCase
• catalytic unit: ___ subunits
organized into ___ trimers
• regulatory unit: ___
subunits organized into
___ dimers
• Catalytic subunits can be
separated from regulatory
subunits by a compound that
reacts with _____________,
(p-hydroxymercuribenzoate)
Allosteric Enzymes (Cont’d)
• Two types of allosteric enzyme systems exist
Note: for an allosteric enzyme, the substrate
concentration at one-half Vmax is called the K0.5
• _________________: an enzyme for which an
inhibitor or activator alters K0.5
• _________________: an enzyme for which an
inhibitor or activator alters Vmax but not K0.5
Allosteric Enzymes (Cont’d)
• The key to allosteric behavior is the existence of _________
____________ for the 4°structure of the enzyme
• ___________________ effector: a substance that
modifies the 4° structure of an allosteric enzyme
• __________ effects: allosteric interactions that occur
when several identical molecules are bound to the
protein; e.g., the binding of aspartate to ATCase
• __________________ effects: allosteric interactions
that occur when different substances are bound to the
protein; e.g., inhibition of ATCase by CTP and
activation by ATP
The Concerted Model
• Wyman, Monod, and Changeux - 1965
• The enzyme has two conformations
• __________ binds substrate tightly; the _______ form
• __________ binds substrate less tightly; ______ form
• in the absence of substrate, most enzyme molecules
are in the __________ __________ form
• the presence of substrate shifts the equilibrium from
the __________ form to the __________ form
• in changing from T to R and vice versa, all subunits
change conformation _______________; all changes
are ______________
Concerted Model (Cont’d)
• A model represented by a protein having two conformations
• Active (R) form - Relaxed binds substrate tightly,
• Inactive (T) form - Tight (taut) binds substrate less tightly, both
change from T to R at the same time
• Also called the __________ model
• Substrate binding shifts equilibrium to the relaxed state.
Any unbound R is removed KR<KT
Ratio of dissociation constants is called c
Monod-Wyman-Changeaux
model
Concerted Model (Cont’d)
• The model explains the _________________ effects
• Higher L means higher favorability of _____________
• Higher c means higher affinity between S and R form,
more ________________ as well.
Concerted Model (Cont’d)
• An allosteric activator (A) binds to and _____________ the
R (active) form
• An allosteric inhibitor (I) binds to and ______________ the
T (inactive) form
• Effect of
binding
activators
and inhibitors
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, as per the induced-fit model
of substrate binding
• sequential model represents cooperativity
Sequential Model (Cont’d)
Sequential model for cooperative binding of substrate
to an allosteric enzyme
• R form is favored by __________________ activator
• Allosteric inhibition also occurs by the ____________
mechanism
• Unique feature of Sequential Model of behavior:
_____________ __________________ - Induced
conformational changes that make the enzyme less
likely to bind more molecules of the same type.
Sequential Model (Cont’d)
• Sequential Model:
Control of Enzyme Activity via Phosphorylation
• The side chain -OH groups
of Ser, Thr, and Tyr can
form __________________
• Phosphorylation by ATP can
convert an _____________
_____________ into an
__________ ___________
• __________ ____________
is a common example
Membrane Transport
Source of PO43- is ______________
• When ATP is hydrolyzed, energy released that drives other
energetically unfavorable reactions to take place
• PO43- is donated to residue in protein by protein ____________
Zymogens
• ______________: Inactive enzyme precursor, cleavage of
one or more covalent bonds transforms it into active enzyme
• Chymotrypsinogen
• synthesized and stored in the pancreas
• a single polypeptide chain of 245 amino acid residues
cross linked by 5 _____________________ bonds
• when secreted into the small intestine, the digestive
enzyme trypsin cleaves a 15 unit polypeptide from the
N-terminal end to give __________________________
Activation of chymotrypsin
• Activation of chymotrypsinogen by proteolysis
Chymotrypsin
• A15-unit polypeptide remains bound to -chymotrypsin by a
________________________________
 -chymotrypsin catalyzes the hydrolysis of 2 dipeptide
fragments to give ___________________________
 -chymotrypsin consists of 3 polypeptide chains joined by 2
of the 5 original disulfide bonds
• changes in 1°structure that accompany the change from
chymotrypsinogen to -chymotrypsin result in changes
in ____________________________________ as well.
 -chymotrypsin is enzymatically ___________ because of its
2°- and 3°structure, just as chymotrypsinogen was
________ because of its 2°- and 3°structure
The Active Site
Important questions to ask about enzyme mode of action:
• Which amino acid residues on an enzyme are in the active site
and catalyze the reaction?
• What is the spatial relationship of the essential amino acids
residues in the active site?
• What is the mechanism by which the essential amino acid
residues catalyze the reaction?
• As a model, we consider chymotrypsin, an enzyme of the
digestive system that catalyzes the selective hydrolysis of
peptide bonds in which the carboxyl group is contributed by
Phe or Tyr
Kinetics of Chymotrypsin Reaction
• p-nitrophenyl acetate is
hydrolyzed by
chymotrypsin in 2
stages.
• At the end of stage 1,
the p-nitrophenolate ion
is released.
• At stage 2, acyl-enzyme
intermediate is
hydrolyzed and acetate
(Product) is released…
free enzyme is
regenerated
Chymotrypsin
• Reaction with a model substrate
Chymotrypsin (Cont’d)
• Chymotrypsin is a ___________ _______________
• DIPF inactivates chymotrypsin by reacting with
serine-195, verifying that _____________________
Chymotrypsin (Cont’d)
• H57 also critical for
__________________
• Can be chemically
__________ by TPCK
N-tosylamido-L-phenylethyl
chloromethyl ketone
Chymotrypsin (Cont’d)
• Because Ser-195 and His-57 are required for activity,
they must be ________________________________
• Results of x-ray crystallography show the definite
___________________________________________
• In addition to His-57 and Ser-195, Asp-102 is also
involved in catalysis at the active site
• The folding of the chymotrypsin backbone, mostly in
_________________________________________,
positions the essential amino acids around the active-site
pocket
Chymotrypsin (Cont’d)
The active site of
chymotrypsin shows
proximity of 2 reactive aa
Mechanism of Action of Critical Amino Acids in
Chymotrypsin
• Serine oxygen is nucleophile
• Attacks carbonyl group of peptide bond
Catalytic Mechanisms
General acid-base catalysis: depends on __________
____________________________________________
• Nucleophilic substitution catalysts - ___________
_____________ atom attacks ______________ atom.
• same type of chemistry can occur at enzyme active
site: SN1, SN2
Catalytic Mechanisms (Cont’d)
• Lewis acid/base reactions
• Lewis acid: an electron pair __________________
• Lewis base: an electron pair __________________
• Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential
components of many enzymes (metal ion catalysts)
• carboxypeptidase A requires Zn2+ for activity
Catalytic Mechanisms (Cont’d)
Zn2+ of _____________
is complexed with:
• The imidazole side
chains of His-69 and
His-196 and the
carboxylate side
chain of Glu-72
Activates the carbonyl
group for nucleophilic
acyl substitution
Enzyme Specificity
• ___________________ specificity: catalyzes the reaction of
one unique substrate to a particular product
• _________________ specificity: catalyzes the reaction of
structurally related substrates to give structurally related
products
• ___________________: catalyzes a reaction in which one
stereoisomer is reacted or formed in preference to all others
that might be reacted or formed
• example: hydration of a cis alkene (but not its trans
isomer) to give an R alcohol (but not the S alcohol)
• Review Cahn–Ingold–Prelog priority rules (R/S) at Wikipedia.
Asymmetric binding
Enzymes can be
_____________________
(Specificity where optical
activity may play a role)
Binding sites on enzymes
must be ______________
Active Sites and Transition States
• Enzyme catalysis
• an enzyme provides an alternative pathway with a lower Ea
• the transition state often has a shape different than the
substrate(s) or the product(s)
• “True nature” of transition state is a species intermediate in
structure between substrate and product.
• Transition state analog: similarly shaped to the 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)
Coenzymes
• Coenzyme: a __________________ that takes part in an
enzymatic reaction and is regenerated 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 biological
redox rxns
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
• Reduction-oxidation occurs on _________________
B6 Vitamins
• The B6 vitamins are coenzymes involved in _______________
____________ ___________ from one molecule to another.
• Important in ______________ _____________ biosynthesis
Pyridoxal Phosphate
• Pyridoxal and pyridoxamine phosphates are involved in
the transfer of __________ ____________ in a reaction
called ________________________
Figure 7.21 p. 197