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Paul D. Adams University of Arkansas
Mary K. Campbell
Shawn O. Farrellhttp://academic.cengage.com/chemistry/campbell
Chapter SevenThe Behavior of Proteins:
Enzymes, Mechanisms, and Control
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Allosteric Enzymes
Allosteric: Greek allo+ steric, other shape
Allosteric enzyme: an oligomer whose biological activity is affected byother substances binding to it
these substances change the enzymes activity by altering the
conformation(s) of its 4structure
Allosteric effector: a substance that modifies the behavior of an allosteric
enzyme; may be an
allosteric inhibitor
allosteric activator
Aspartate transcarbamoylase (ATCase)
feedback inhibition
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Feedback Inhibition
Formation of product
inhibits its continuedproduction
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ATCase
Rate of ATCase catalysis vs
substrate concentration
Sigmoidal shape of curve describes
allosteric behavior
ATCase catalysis in presenceof CTP; ATP
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ATCase (Contd)
Organization of ATCase
catalytic unit: 6 subunitsorganized into 2 trimers
regulatory unit: 6 subunitsorganized into 3 dimers
Catalytic subunits can beseparated from regulatorysubunits by a compound thatreacts with cysteine (p-hydroxymercuribenzoate)
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Allosteric Enzymes (Contd)
Two types of allosteric enzyme systems exist
Note: for an allosteric enzyme, the substrateconcentration at one-half Vmax is called the K0.5
K system: an enzyme for which an inhibitor oractivators alters K0.5
V system: an enzyme for which an inhibitor oractivator alters Vmax but not K0.5
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Allosteric Enzymes (Contd)
The key to allosteric behavior is the existence of multiple
forms for the 4structure of the enzyme allosteric effector: a substance that modifies the 4
structure of an allosteric enzyme
homotropic effects: allosteric interactions that occur
when several identical molecules are bound to theprotein; e.g., the binding of aspartate to ATCase
heterotropic effects: allosteric interactions that occurwhen different substances are bound to the protein;e.g., inhibition of ATCase by CTP and activation byATP
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The Concerted Model
Wyman, Monod, and Changeux - 1965
The enzyme has two conformations
R (relaxed): binds substrate tightly; the active form
T (tight or taut): binds substrate less tightly; the
inactive form in the absence of substrate, most enzyme molecules
are in the T (inactive) form
the presence of substrate shifts the equilibrium from
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 areconcerted
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Concerted Model (Contd)
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 atthe same time
Also called the concerted model
Substrate binding shifts equilib. To the relaxed state.
Any unbound R is removed KR
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Concerted Model (Contd)
The model explains the sigmoidal effects
Higher L means higher favorability of free T form
Higher c means higher affinity between S and R form,more sigmoidal as well.
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Concerted Model (Contd)
An allosteric activator (A) binds to and stabilizes the R
(active) form An allosteric inhibitor (I) binds to and stabilizes the T
(inactive) form
Effect ofbindingactivatorsand inhibitors
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Sequential Model (Contd)
Main Feature of Model:
the binding of substrate induces a conformationalchange from the T form to the R form
the change in conformation is induced by the fit of thesubstrate to the enzyme, as per the induced-fit modelof substrate binding
sequential model represents cooperativity
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Sequential Model (Contd)
Sequential model for cooperative binding of substrate to an allosteric enzyme
R form is favored by allosteric activator
Allosteric inhibition also occurs by the induced-fit mechanism
Unique feature of Sequential Model of behavior:
Negative cooperativity- Induced conformational changes that make the enzyme
less likely to bind more molecules of the same type.
Sequential Model:
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Control of Enzyme Activity via Phosphorylation
The side chain -OH groups
of Ser, Thr, and Tyr canform phosphate esters
Phosphorylation by ATP can
convert an inactiveprecursor into an activeenzyme
Membrane transport is acommon example
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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
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Zymogens
Zymogen: Inactive precursor of an enzyme where cleavage
of one or more covalent bonds transforms it into the activeenzyme
Chymotrypsinogen
synthesized and stored in the pancreas
a single polypeptide chain of 245 amino acid residuescross linked by five disulfide (-S-S-) bonds
when secreted into the small intestine, the digestiveenzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give -chymotrypsin
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Activation of chymotrypsin
Activation of chymotrypsinogen by proteolysis
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Chymotrypsin
A15-unit polypeptide remains bound to -chymotrypsin by
a single disulfide bond -chymotrypsin catalyzes the hydrolysis of two dipeptide
fragments to give -chymotrypsin
-chymotrypsin consists of three polypeptide chains joined
by two of the five original disulfide bonds changes in 1structure that accompany the change from
chymotrypsinogen to -chymotrypsin result in changes in
2- and 3structure as well.
-chymotrypsin is enzymatically active because of its 2
-and 3structure, just as chymotrypsinogen was inactivebecause of its 2- and 3structure
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The Active Site
Some important questions to ask about enzyme mode of action:
Which amino acid residues on an enzyme are in the active siteand catalyze the reaction?
What is the spatial relationship of the essential amino acidsresidues in the active site?
What is the mechanism by which the essential amino acidresidues catalyze the reaction?
As a model, we consider chymotrypsin, an enzyme of thedigestive system that catalyzes the selective hydrolysis ofpeptide bonds in which the carboxyl group is contributed byPhe or Tyr
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Kinetics of Chymotrypsin Reaction
p-nitrophenyl acetate is
hydrolyzed bychymotrypsin in 2stages.
At the end of stage 1,
the p-nitrophenolate ionis released.
At stage 2, acyl-enzymeintermediate ishydrolyzed and acetate(Product) isreleasedfree enzyme
is regenerated
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Chymotrypsin
Reaction with a model substrate
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Chymotrypsin (Contd)
Chymotrypsin is a serine protease
DIPF inactivates chymotrypsin by reacting withserine-195, verifying that this residue is at the active
site
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Chymotrypsin (Contd)
H57 also critical for
activation of enzyme
Can be chemically
labeled by TPCK
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Chymotrypsin (Contd)
Because Ser-195 and His-57 are required for activity,
they must be close to each other in the active site
Results of x-ray crystallography show the definitearrangement of amino acids at the active site
In addition to His-57 and Ser-195, Asp-102 is alsoinvolved in catalysis at the active site
The folding of the chymotrypsin backbone, mostly inantiparallel pleated sheet array, positions the essential aminoacids around the active-site pocket
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Chymotrypsin (Contd)
The active site of
chymotrypsin showsproximity of 2 reactivea.a.
M h i f A ti f C iti l A i A id i
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Mechanism of Action of Critical Amino Acids inChymotrypsin
Serine oxygen is nucleophile
Attacks carbonyl group of peptide bond
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Catalytic Mechanisms
General acid-base catalysis: depends on donation
and acceptance of protons (proton transfer reactions)
Nucleophilic substitution catalysts- Nucleophilicelectron-rich atom attacks electron deficient atom.
same type of chemistry can occur at active site of
enzyme: SN1, SN2
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Catalytic Mechanisms (Contd)
Lewis acid/base reactions
Lewis acid: an electron pair acceptor
Lewis base: an electron pair donor
Lewis acids such as Mn2+, Mg2+, and Zn2+ are essentialcomponents of many enzymes (metal ion catalysts)
carboxypeptidase A requires Zn2+ for activity
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Catalytic Mechanisms (Contd)
Zn2+ of
carboxypeptidase iscomplexed with:
The imidazole sidechains of His-69 andHis-196 and the
carboxylate sidechain of Glu-72
Activates the
carbonyl group fornucleophilic acylsubstitution
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Enzyme Specificity
Absolute specificity: catalyzes the reaction of one unique
substrate to a particular product
Relative specificity: catalyzes the reaction of structurallyrelated substrates to give structurally related products
Stereospecificity: catalyzes a reaction in which onestereoisomer is reacted or formed in preference to all othersthat might be reacted or formed
example: hydration of a cis alkene (but not its transisomer) to give an R alcohol (but not the S alcohol)
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Asymmetric binding
Enzymes can be
stereospecific(Specificity whereoptical activity may paya role)
Binding sites on enzymes
must be asymmetric
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Active Sites and Transition States
Enzyme catalysis
an enzyme provides an alternative pathway with a loweractivation energy
the transition state often has a different shape than either thesubstrate(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. Transition state analog: a substance whose shape mimics that of atransition 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)
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Coenzymes
Coenzyme: a nonprotein substance 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).
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NAD+/NADH
Nicotinamide adenine
dinucleotide (NAD+
) is usedin many redox reactions inbiology.
Contains:1) nicotinamide ring
2) Adenine ring
3) 2 sugar-phosphate groups
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NAD+/NADH (Contd)
NAD+ is a two-electron oxidizing agent, and is
reduced to NADH
Nicotinamide ring is where reduction-oxidation
occurs
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B6 Vitamins
The B6 vitamins are coenzymes involved in amino group
transfer from one molecule to another. Important in amino acid biosynthesis
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Pyridoxal Phosphate
Pyridoxal and pyridoxamine phosphates are involved in
the transfer of amino groups in a reaction calledtransamination
Figure 7.21 p. 197