Mechanism of Enzyme Action Flashcards
a species inter-mediate in structure
between S and P
Transition State
TRUE OR FALSE
the enzymatic rate enhancement is
approximately not equal to the ratio of
the dissociation constants of the E-S
and E- transition-state complexes, at
least when E is saturated with S
False (Equal)
TRUE OR FALSE
the E must stabilize the Substrate complex, EX‡, more than it stabilizes the Transition State Complex, ES
False (transition state complex, substrate complex)
ensures the favorable formation of
the ES complex
(triangle)Gb (Intrinsic binding)
Gb is partially compensated by
Blank due to the binding of E
and S (TS) and by Blank
(Gd) by strain, distortion, desolvation,
and similar effects
entropy loss, destabilization of ES
TRUE OR FALSE
the smaller the difference in
energies between ES and EX‡, the
Slower the E-catalyzed reaction
False (faster)
Blank of the substrate
deepens the energy well of the ES
complex and actually lowers the
rate of the reaction
tight binding
can involve structural strain, desolvation,
or electrostatic effects
destabilization of the ES complex
a consequence of the fact that the E
is designed to bind the transition
state more strongly than the S
Destabilization by strain or distortion
E bind the transition-state structure
more tightly than the Blank
S (or the P)
How Destabilization of the E-S
Complex Affect Enzyme Catalysis?
Destabilization of the enzyme-substrate (E-S) complex enhances enzyme catalysis by preventing overly stable binding. When the E-S complex is less stable, the energy difference between it and the transition state is reduced, lowering the activation energy required for the reaction. This promotes the formation of the transition state and increases the overall reaction rate. By binding the substrate with moderate affinity, the enzyme facilitates a smoother transition to the enzyme-transition state complex, improving catalytic efficiency.
How is it that the transition state X‡
is stabilized more than S at the E
active site?
The favorable interactions between the substrate (S) and the amino acid (AA) residues on the enzyme (E) contribute to the intrinsic binding energy, #Gb..
TRUE OR FALSE
solvation of charged groups on a
substrate in solution releases
energy, making the charged
substrate more unstable
False (Stable)
if the charge on the S is diminished or lost in the course of reaction, electrostatic destabilization can result
in Blank
rate acceleration
when a S enters the active site,
charged groups may be forced to
interact (unfavorably) with charges of like sign, resulting in Blank
electrostatic destabilization
- a “moving target”
- exists for about 10-14 to 10-13 s
transition state
electrostatic destabilization of a substrate may arise from Blank of like charges in the active site
juxtaposition
if such charge repulsion is relieved in the course of the reaction, electrostatic destabilization can
result in a Blank
rate increase
pyrrole-2-carboxylate binds to pro racemase Blank more tightly
than L-proline, the normal S
160 x
TRUE OR FALSE
dihydroxyacetone phosphate binds 40,000 times more tightly to
yeast aldolase than the substrate Phosphoglycolohydroxamate.
False (Phosphoglycolohydroxamate binds 40,000 times more tightly to
yeast aldolase than the substrate dihydroxyacetone phosphate.)
Blank of purine ribonucleoside has been estimated to bind to adenosine deaminase with a KI of 3 x 10-13 M
1,6-hydrate
What are the Mechanism of Catalysis ?
- Near-Attack Conformations (NACs)
- Protein Motions
- Covalent Catalysis
- General acid-base catalysis
- Low-Barrier Hydrogen Bonds (LBHB)
- Quantum Mechanical Tunneling in Electron and Proton Transfers
- Metal ion Catalysis
- Noncatalytic Residues
- the reacting atoms are in van der
Waals contact and at an angle
resembling the bond to be formed
in the transition state
Near-Attack Conformations (NACs)
in the absence of an E, potential
reactant molecules adopt a NAC
only about Blank of the time
0.0001%
NACs have been shown to form in E
active sites from Blank to blank of the
time
1% to 70%
- proteins are constantly moving
- bonds vibrate, side chains bend and
rotate, backbone loops wiggle and
sway, and whole domains move
with respect to each other - E depend on such motions to
initiate and direct catalytic events
Protein motions
Protein motions may support catalysis
in several ways. Active site
conformation changes can (5)
- assist substrate binding
- bring catalytic groups into position
around a substrate - induce formation of a NAC
- assist in bond making and bond
breaking - facilitate conversion of S to P
acceptor group on the E must be a
better attacking group than Y and a
better leaving group than X
Covalent catalysis
formation of covalent bonds
between E and S
BX + Y → BY + X
enzymatic version
BX + Enz → E:B + X + Y → Enz + BY
covalent intermediate
readily attack electrophilic centers of S,
forming covalently bonded E-S
intermediates
nucleophilic centers for catalysis
what are the side chains of AA in proteins?
✓ amines
✓ carboxylates
✓ aryl and alkyl hydroxyls
✓ imidazoles
✓ thiol groups
- a proton is transferred in the
transition state - may increase reaction rates 10- to
100-fold
general acid-base catalysis
is often the most effective
general acid or base because the pKa
of the histidine side chain is near 7
histidine
- when the barrier-to-hydrogen
exchange has dropped to the point
that it is at or below the zero point
energy level of hydrogen
Low-Barrier Hydrogen Bonds (LBHB)
the stabilization energy of LBHBs
may approach Blank in the gas
phase and Blank or more in
solution
100 kJ/mol, 60 kJ/mol
TRUE OR FALSE
as the two pKa values diverge, the
stabilization energy of the LBHB is
increased
False (decreased)
a Blank in an enzyme ground
state may become an LBHB in a
transient intermediate, or even in the
transition state for the reaction
weak H bond
- if an atom or electron is transferred in a
chemical reaction from one site to
another across an activation barrier,
there is a finite probability that the
particle will appear (as part of theP) on
the other side of the energy barrier,
even though it cannot achieve sufficient
energy to reach the transition state
quantum theory
What are the 2 Metal Ion Catalysis?
- Metalloenzyme
- Metal Activated
- if the E binds the metal very tightly
or requires the metal ion to
maintain its stable, native state
metalloenzyme
- E that bind metal ions more weakly,
perhaps only during the catalytic
cycle
metal activated
is an endoprotease (it cleaves polypeptides in the middle of the chain) with a catalytic Zn2+
ion in the active site
Thermolysin
Role of a metal includes act as Blank, stabilizing the increased e- density
or (-) charge that can develop
during rxn
electrophilic catalysts
coordination to a metal ion can
increase the Blank of a nucleophile
w/ an ionizable proton
acidity
- raising or lowering catalytic residue
pKa values through electrostatic or
hydrophobic interactions - orientation of catalytic residues
- charge stabilization
- proton transfers via hydrogen
tunneling
Noncatalytic residues
What are the 2 roles of metal?
- Act as electrophilic catalysts, stabilizing the increased e- density or (-) charge that can develop during reaction.
- Provide a powerful nucleophile at neutral pH.
- a class of proteolytic enzymes
whose catalytic mechanism is based
on an active-site serine residue
Serine Proteases
What are the 3 digestive enzymes?
- Trypsin
- Chymotrypsin
- Elastase
Blood-clotting enzyme
Thrombin
Bacterial Protease
Subtilisin
Breaks down the fibrin polymers of blood clots
Plasmin
- cleaves the proenzyme
plasminogen, yielding plasmin - minimizes the harmful
consequences of a heart attack, if
administered to a patient w/in 30
min of onset
tissue plasminogen activator (TPA)
- a serine esterase and is related
mechanistically to the serine
proteases - degrades the neurotransmitter
acetylcholine in the synaptic cleft
between neurons
Acetylcholinesterase (not a protease)
- cleaves peptides on the carbonyl
side of the basic AAs, arg or lysine
trypsin
- cleaves peptides on the carbonyl
side of small, neutral residues
elastase
- cleaves on the carbonyl side of
aromatic residues, phe and tyr
chymotrypsin
What are the 3 polar residues—— at the active site
His57, Asp102, Ser195
Blank (red) is flanked by
Blank (gold) and by and
Blank (green)
His57
Asp102
Ser195
the catalytic site is filled by
a peptide segment of Blank
Eglin
is actually a depression on the surface of the
enzyme, with a pocket that the
enzyme uses to identify the residue
for which it is specific
the active site
has a pocket surrounded by hydrophobic
residues and large enough to
accommodate an aromatic side
chain
chymotrypsin
the pocket in trypsin has a Blank at its bottom, facilitating the binding of positively
charged Blank and Blank residues
negative charge (Asp189)
arginine and lysine
has a shallow pocket with
bulky thr and val residues at the
opening; only small, nonbulky
residues can be accommodated in
its pocket
elastase
in the active sites of all these
enzymes, the backbone of the
peptide substrate is hydrogen bonded
in antiparallel fashion to residues Blank to Blank and bent so that the peptide
bond to be cleaved is bound close to
Blank to Blank
215 to 219
His57 and Ser195
the serine protease mechanism relies
in part on a low-barrier hydrogen
bond between Blank and Blank
Asp102 and His57
- produced by mammals, fungi and
higher plants - active at acidic (or sometimes
neutral) pH
Aspartic Proteases
Digestion of dietary protein
Pepsin
Digestion of dietary protein
Chymosin
Lysosomal digestion of proteins
Cathepsin D
Processing of AIDS virus protein
HIV-protease
Conversion of angiotensinogen to angiotensin 1; regulation of blood pressure
Renin
- a hormone that stimulates smooth muscle contraction and
reduces excretion of salts and fluid
angiotensoin
- display a variety of S specificities,
but normally they are most active in
the cleavage of peptide bonds
between two hydrophobic AA
residues
aspartic proteases
Aspartic proteases is composed of Blank to Blank AA residues
323 to 340
*2 homologous domains that fold to
produce a tertiary structure composed of 2
similar lobes, with approximate 2-fold
symmetry
aspartic protease polypeptides
each of these lobes or domains consists of
Blank and Blank
2 β-sheets and 2 short a-helices
the 2 domains are bridged and connected
by a Blank, Blank
6-stranded, antiparallel β-sheet
the active site is a deep and extended
cleft, formed by the Blank and large enough to accommodate about Blank AA residues
2 juxtaposed domains, 7
- causative viral agent of AIDS
human immunodeficiency virus (HIV-1)
- cleaves the polyprotein products of
the HIV-1 genome, producing several
proteins necessary for viral growth
and cellular infection - cleaves several different peptide
linkages
HIV-1 protease
- a remarkable viral imitation of
mammalian aspartic proteases - a dimer of identical subunits that
mimics the 2-lobed monomeric
structure of pepsin and other aspartic
proteases
HIV-1 protease
HIV-1 protease cleaves between the Blank and Blank residues of the sequence Ser-Gln-Asn-Tyr-Pro-Ile-Val, which joins the Blank and Blank HIV-1 proteins
Tyr and Pro
p17 and p24
How many residue polypeptides that are
homologous with the individual
domains of the monomeric protease?
99
structures determined by X-ray
diffraction studies reveal that the
active site of HIV-1 protease is formed
at the interface of the homodimer and
consists of 2 asp residues, designated
Blank and Blank, one contributed by
each subunit
Asp25 and Asp259
- IN EQUILIBRIUM STATE WHAT HAPPENED TO PRODUCT AND SUBSTRATE?
-At equilibrium in an enzyme-catalyzed reaction, the rates of the forward and reverse reactions are equal, so there’s no net change in the concentrations of substrate, product, or enzyme. The enzyme constantly binds and releases the substrate, maintaining a steady state with stable levels of substrate and product. Although molecules continue to interact, the overall concentrations remain balanced.
- WHY ENZYMES MUST BE DESTROYED?
- Their degradation helps regulate metabolic pathways, ensuring that products are neither overproduced nor depleted. Enzymes, like all proteins, have a limited lifespan and are recycled to maintain cellular health by removing damaged or misfolded ones. Environmental changes, such as shifts in pH or temperature, can also denature enzymes, preventing unwanted reactions. This natural turnover allows cells to produce fresh enzymes as needed, ensuring efficiency and proper control of cellular processes, such as growth and signaling.
in which a substrate (S) is converted to a
product (P) can be pictured as
involving a transition state
chemical reactions
- stable molecules that are chemically and structurally similar to the transition state
transition-state analogs
