Transcript 11 Enzymes - School of Chemistry and Biochemistry

revised 10/31/2013
Biochemistry I
Dr. Loren Williams
Chapter 11
Enzymatic Catalysis
Copyright © 2008 by John Wiley & Sons, Inc.
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Chapter 11 Opener
high reaction rates,
controllable reaction rates
high specificity,
mild conditions
Table 11-1
Table 11-2
Enzymes bind to specific substrates,
via non-covalent interactions
complementarity of surfaces, and interactions,
sometimes ‘lock and key’
sometimes ‘induced fit’
with stereospecificity
generally but not always with chemical (geometric) specificity
peptidases versus esterases
Figure 11-1
Prochirality:
A tetrahedral carbon that can be
converted to a chiral carbon by
changing any one of its attached
groups is ‘prochiral’. All the
substitutuents of a prochiral carbon
are distinguishable in a chiral
environment (in the vicinity of any
biological macromolecule).
The brown and the blue functional
groups behave identically in an
achiral environment.
In a chiral environment the brown
and the blue functional groups are
different and can be distinguished.
These two can be distinguished by
an enzyme: they occupy different
regions in three-dimensional space.
Prochirality is an important concept
in biological chemistry and was
discovered by biochemists.
Page 325
A prochiral molecule in chiral
environment - is chiral.
Prochiral:
potentially chiral
nearly asymmetric
Figure 11-2
This molecule is prochiral, the two faces are different, only
when it is in a chiral environment. Otherwise, they are the
same.
Some enzymes are not too specific (this is unusual).
Page 326
Figure 11-3
NAD
or NADP
NADH
or NADPH
Schematic structures of distinct identifying domains in Nox isoform catalytic subunits.
NADPH Oxidases: Schematic structures of distinct identifying domains in Nox isoform
catalytic subunits. A: Nox2 and Nox2-like isoforms (Nox1, 3, and 4) present a basic
structure of 6 transmembrane domains containing two heme groups and COOH-terminal
FAD and NADPH domains. B: in addition to those domains, Nox5 possesses Ca2+binding EF motifs in the NH2-terminal region. C: dual oxidasess, in addition to Ca2+binding EF motifs, possess an NH2-terminal peroxidase-like domain.
Frazziano G et al. Am J Physiol Heart Circ Physiol
2012;302:H2166-H2177
reduced
Page 327
oxidized
oxidized
reduced
Figure 11-5a
Figure 11-5b
Figure 11-6
Figure 11-7
Relationships between
rate constants
equilibrium constants
activation free energy
free energy of reaction
DG°rxn=-RTlnK
DG°rxn= DH°rxn-TDS°rxn
K = kr/ff ;
DG°rxn = DG°‡rDG°‡f
The highest energy species (most unstable species)
along the reaction coordinate.
Page 330
uncatalyzed
acid catalyzed
base catalyzed
Figure 11-8
Figure 11-8a
Figure 11-8b
Figure 11-8c
Effect of pH on catalytic
activity.
This profile suggests a
histidine is involved in the
mechanism.
Box 11-1
•acids & bases
•covalent intermediates
•metal ions
•proximity and orientation
(VVP calls this section ‘mechanisms of catalysis, that
seems a bit illogical. These are components of
mechanisms.)
an acid and a base
Figure 11-9
E + S ⇌ ES ⇌ EI ⇌ EP ⇌ E + P
Figure 11-10
Figure 11-10 part 1
Figure 11-10 part 2
enzyme
nucleophile
Page 334
uncatalyzed reaction
(nucleophile on enzyme)
catalyzed reaction
Figure 11-11
Figure 11-12
Carbonic Anhydrase
The carbonic anhydrase,
o a metalloenzyme (contains a Zn2+)
o catalyzes the conversion of
bicarbonate and a proton to carbon
dioxide and water
o also catalyzes the the reverse:
conversion of carbon dioxide and
water to bicarbonate and a proton
o is a very efficient enzyme
o causes carbonated drinks to rapidly
degas in your mouth
the Zn2+ is square planer
is coordinated by three His
plus one water molecule
the water molecule is polarized
by the Zn and is highly acidic
Figure 11-13a
Figure 11-13b
(i) Proximity increases reaction rates
Slower
Faster
Page 336
(ii) Orientation increases reaction rates:
Correct, fixed orientation increases reaction rates.
For example in an SN2 reaction, the reaction is fastest if
the attacking nucleophile approaches the electrophilic C
along the line of the leaving group bond.
Figure 11-14
The transition state can be stabilized by mechanically distorting
the substrate to a non-ground state conformation that is close to
the transition state conformation.
This example shows how a non-enzymatic transition state can
be stabilized (Big R lowers DG‡)
Page 338
E + S ⇌ ES ⇌ EP ⇌ E + P
Figure 11-15
I don’t’ get this graph, which is
intended to relate to preferential
binding of the enzyme to the
transition state. In fact, all the
elements of catalysis discussed in
this section (acid base, metals,
proximity and orientation) will lower
DG‡ and will contribute to a graph
shape like this. The double headed
arrow is the binding free energy of
the substrate to the enzyme. To be
most specifically related to binding
to the transition state this graph
should have H (enthalpy) on the
horizontal axis and should explain
the the binding energy can be
converted to mechanical distortion
of the substrate toward the transition
state.
‡
‡
Lysozyme
Lysozyme breaks up peptidoglycans, which form the cell walls
of bacteria. Lysozyme hydrolyzes the glycosidic bond that
connects the 1-oxygen of N-acetylmuramic acid with the 4carbon of N-acetylglucosamine. Lysozyme conferes protection
from bacterial infection. For example the membrane covering
the eye is protected by secreted lysozyme and defensin.
The 3D structure of chicken egg-white lysozyme was
determined to 2 Å resolution by X-ray diffraction (Phillips,
1965). Lysozyme was the second protein structure determined
(after myoglobin, Perutz & Kendrew) and was the first enzyme
whose structure was solved. Lysozyme was therefore the first
enzyme for which the detailed mechanism was known.
Peptidogycan
are stained by Gram
staining
are not stained by
Gram staining
Plant cell walls are made from cellulose. Bacterial cell walls are made from
peptidoglycan.
Gram-positive cell walls have a high amount of peptidoglycan in their cell walls and
lack outer membranes, unlike in Gram-negative bacteria.
Figure 8-16a
Gram negative bacteria contain an outer membrane composed of
lipopolysaccharide which contains porins (hole-making proteins). The
space between the peptidoglycan and the outer membrane is called the
periplasmic space.
Peptidoglycan - carbohydrates + peptides
carbohydrates: alternating N-acetylglucosamine (NAM) and Nacetylmuramic acid (NAG) with β-(1,4) linkages.
Peptides - oligopeptides (3-5 AA long) linked to N-acetylmuramic
acids, and cross-linked to other oligopeptides (pentaglycine)
The peptide chain cross-links to the peptide chain of another strand forming
the 3D mesh-like layer.
Peptidoglycan serves a structural role in the bacterial cell wall, giving
structural strength, as well as counteracting the osmotic pressure of the
cytoplasm.
NAG
N-Acetyl-D-Glucosamine
GlcNAc
NAM
N-acetylmuramic acid
Figure 8-17a
Penicillin was discovered by Alexander Fleming in 1928. He showed
the fungus Penicillium notatum can exude a substance with
antibiotic properties, which he dubbed penicillin. In one of the best
examples of the power of basic research, Fleming's 'serendipitous'
discovery (from a discarded, contaminated Petri dish) has saved
100’s of millions of lives. Penicillin prevents formation of
peptidoglycan cross-links by inhibiting the the enzyme (DDtranspeptidase).
D
N-acetylmuramic acid
Figure 11-16
E
N-acetylglucosamine
Figure 11-17
Figure 11-19
Identifying the cleaved bond and the products: retention of
configuration.
18-O at C1 or C4?
Page 343
oxonium ion-carbocation:
trigonal planer intermediate
non-enzymatic cleavage
Figure 11-20
Figure 11-21
The enzyme binds to the substrate and some of the binding energy is
used to distort the D-ring to the half chair. Therefore the energy of
binding is used to stabilize the transition state.
(ignore the electron flow arrows in this slide, that comes later)
D
Figure 11-21 part 1
Proton transfer to the bridging O1
atom. Now the electrons flow.
cleavage
trigonal planer intermediate
Figure 11-21 part 2
D
D
nucleophilic
attack by Asp
52
tetrahedral intermediate,
covalent E-S intermediate
Figure 11-21 part 3
D
D
D
Ignore the electron flow on this panel
Water replaces the
E ring in the active site.
Figure 11-21 part 4
5) Then, product release.
4) And ASP35 – substrate bond
cleaves
3) And, base-catalysis (Glu35 accepts
a proton from the water).
2) Then, nucleophilic attack
by water at the C1
of the D ring
1) After water replaces the
E ring in the active site (previous
slide_.
Figure 11-21 part 5
Figure 11-22
E35Q
(no acid/base catalysis)
NAG2FGLcF
F magenta (stabilize carbocation_
Figure 11-23
Serine proteases
Cut peptide bonds
Are grouped into clans that share structural similarities (homology)
chymotrypsin-like,
the subtilisin-like,
the alpha/beta hydrolase,
signal peptidase clans.
Ancestral serine proteases in mammels were digestive enzymes that
evolved by gene duplication and now function in blood clotting, the
immune system, and inflammation.
Serine proteases are paired with serine protease inhibitors.
not covered, fall 2013
Page 348
not covered, fall 2013
Figure 11-24
not covered, fall 2013
The nucleophilic serine
can be identified by
chemical labeling.
Page 348
Acetylcholine is aneurotransmitter.
Accumulation of acetylcholine causes continuous stimulation of
the muscles, glands, and central nervous system.
DIPF, Sarin and VX nerve gas are acetylcholine esterase
inhibitors.
Box 11-3a
Box 11-3c
acetylcholine esterase inhibitors
Box 11-3b
Figure 11-25
Figure 11-26
Specificity pockets
Figure 11-27
Not covered, Fall 2013
Figure 11-28
Figure 11-29
Figure 11-29 part 1
Figure 11-29 part 2
Figure 11-29 part 3
Figure 11-29 part 4
Figure 11-29 part 5
Figure 11-30a
Figure 11-30b
BPTI:
bovine pancreatic
trypsin inhibitor
BPTI is a member of the
protein family of Kunitztype serine protease
inhibitors. Its primary
function seems to be
inhibition of trypsin in the
pancreas.
Small amounts of
trypsinogen are cleaved
during storage in the
pancreas (not good).
Wikipedia: BPTI is one of the most thoroughly studied proteins by structural
biology methods, experimental and computational dynamics, mutagenesis, and
folding experiments. It was one of the earliest protein crystal structures solved, in
1970 in the laboratory of Robert Huber, and was the first protein to have its
structure determined by NMR spectroscopy. It was first macromolecule of
scientific interest to be simulated using molecular dynamics.
Figure 11-31a
Trypsin
BPTI
Figure 11-31b
elastase, ph 5.0:
Figure 11-32
elastase, ph 9.0, quick freeze:
Figure 11-32a
Figure 11-32b
Trypsin is dangerous and is synthesized as an inactive form called
a zymogen. The zymogen of trypsin is called trypsinogen, which is
found in pancreatic juice, along with amylase, lipase,
chymotrypsinogen, nucleases, etc. Trypsinogen is activated
(converted to trypsin) by cleavage after amino acid 15 by the
protease enteropeptidase. Enteropeptidase is found in the
intestinal mucosa. In addition, Trypsin can cleave trypsinogen to
form trypsin.
Figure 11-33
Coagulation Cascade
Box 11-4a
Factor XIa is a serine protease that is synthesized as a zymogen called factor XI.
Factor XIa activates Factor IX by proteolysis to give Factor IXa.
Factor IXa is a serine protease (synthesized as zymogen Factor IX).
Factor IXa activates Factor X by proteolysis to give Factor Xa
Factor Xa is a serine protease (synthesized as zymogen Factor X).
The activity of factor Xa is enhanced by Factor V.
Factor Xa cleaves prothrombin to form thrombin.
Thrombin is a serine protease (synthesized as zymogen prothrombin).
image from promega
Box 11-4b
Hemophilia A is clotting factor
VIII deficiency (1 in 5,000–10,000
male births.)
Hemophilia B is factor IX
deficiency (1 in about 20,000–
34,000 male births).
Box 11-4c
Protease Classes
Serine proteases
Threonine proteases
Cysteine proteases
Aspartate proteases
Glutamic acid proteases
Metalloproteases
Enzymes that catalyse the hydrolytic cleavage of peptide bonds are called proteases. Proteases fall
into four main mechanistic classes: serine, cysteine, aspartyl and metalloproteases. In the active
sites of serine and cysteine proteases, the eponymous residue is usually paired with a protonwithdrawing group to promote nucleophilic attack on the peptide bond. Aspartyl proteases and
metalloproteases activate a water molecule to serve as the nucleophile, rather than using a
functional group of the enzyme itself. However, the overall process of peptide bond scission is
essentially the same for all protease classes. Soluble serine proteases (a); cysteine proteases (b);
aspartyl proteases (c); and metalloproteases (d). © 2009 Nature Publishing Group