Chapter 11Enzymatic Catalysis

Chapter 11Enzymatic Catalysis

Revised 4/30/2014

Biochemistry IDr. Loren Williams

Biochemistry IDr. Loren Williams

Chapter 11 Opener

Table 11-1

high reaction rates,controllable reaction rates (cooperativity, allosterism)high specificity,mild conditions.

Table 11-2

Figure 11-1

Enzymes bind to specific substrates,via non-covalent interactionscomplementarity of surfaces, and interactions,sometimes ‘lock and key’sometimes ‘induced fit’with stereospecificitygenerally but not always with chemical (geometric) specificitypeptidases versus esterases

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.

Figure 11-2

A prochiral molecule in chiral environment - is chiral.

Prochiral: potentially chiralnearly asymmetric

This molecule is prochiral, the two faces are different, only when it is in a chiral environment. Otherwise, they are the same.

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Some enzymes are not too specific (this is unusual).

Figure 11-3

NADHor NADPH

NADor NADP

Schematic structures of distinct identifying domains in Nox isoform catalytic subunits.

Frazziano G et al. Am J Physiol Heart Circ Physiol 2012;302:H2166-H2177

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 NH

2-terminal region. C: dual oxidasess, in addition to Ca2+-

binding EF motifs, possess an NH2-terminal peroxidase-like domain.

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reducedoxidizedreduced oxidized

Figure 11-5a

Figure 11-5b

Wha

t do

es a

n en

zym

e do

to

this

gra

ph?

Figure 11-6

Figure 11-7

Relationships between rate constants equilibrium constantsactivation free energyfree energy of reaction

G°rxn=-RTlnK

G°rxn= H°

rxn-TS°rxn

 K = kr/ff ; G°rxn = G°‡

r- G°‡f

The highest energy species (most unstable species) along the reaction coordinate.

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Figure 11-8

uncatalyzed

acid catalyzed

base catalyzed

Figure 11-8a

Figure 11-8b

Figure 11-8c

Box 11-1

Effect of pH on catalytic activity.This profile suggests a histidine is involved in the mechanism.

•acids & bases•covalent intermediates•metal ions•proximity and orientation

(VVP calls this section ‘mechanisms of catalysis, that seems a bit illogical. These are components or elements of catalysis. Catalytic mechanisms incorporate one or more of these elements.)

Figure 11-9

an acid and a base

Figure 11-10

E + S ES EI EP E + P⇌ ⇌ ⇌ ⇌

Figure 11-10 part 1

Figure 11-10 part 2

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nucleophile

enzyme

Figure 11-11

uncatalyzed reaction

catalyzed reaction

(nucleophile on enzyme)

Figure 11-12

Figure 11-13a

Carbonic Anhydrase

The carbonic anhydrase, oa metalloenzyme (contains a Zn2+) ocatalyzes the conversion of bicarbonate and a proton to carbon dioxide and wateroalso catalyzes the the reverse: conversion of carbon dioxide and water to bicarbonate and a protonois a very efficient enzymeocauses carbonated drinks to rapidly degas in your mouththe Zn2+ is square planer

is coordinated by three Hisplus one water moleculethe water molecule is polarized by the Zn and is highly acidic

Figure 11-13b

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Faster

(i) Proximity increases reaction rates

Slower

Figure 11-14

(ii) Orientation increases reaction rates:(i.e., correct + fixed orientation increases reaction rates.)For example an SN2 reaction reaction is fastest if the attacking nucleophile approaches the electrophilic C along the line of the leaving group bond, from the opposite side of the C.

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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-group lowers G‡)

Figure 11-15

E + S ES EP E + P⇌ ⇌ ⇌

I don’t’ like 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 G‡ and will contribute to a graph shape like this. The small double headed arrow is the binding free energy of the substrate to the enzyme. I think that to be specifically related to binding to the transition state, this graph should have H (enthalpy) on the vertical axis and should explain that 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 4-carbon 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.

are stained by Gram staining

are not stained by Gram staining

Peptidogycan

Figure 8-16a

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.

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 + peptidescarbohydrates: alternating N-acetylglucosamine (NAM) and N-

acetylmuramic 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.

NAMN-acetylmuramic acid

NAGN-Acetyl-D-GlucosamineGlcNAc

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 (DD-transpeptidase).

Figure 11-16

N-acetylglucosamineN-acetylmuramic acid

D E

Figure 11-17

Figure 11-19

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Identifying the cleaved bond and the products: retention of configuration. 18-O at C1 or C4?

Figure 11-20

non-enzymatic cleavage

oxonium ion-carbocation:trigonal planer intermediate

Figure 11-21

Figure 11-21 part 1

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 2

Proton transfer to the bridging O1 atom. Now the electrons flow.

cleavage

trigonal planer intermediate D D

Figure 11-21 part 3

nucleophilic attack by Asp 52

tetrahedral intermediate,covalent E-S intermediate

D D

D

Figure 11-21 part 4

Water replaces the E ring in the active site.

Ignore the electron flow on this panel

Figure 11-21 part 5

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 C1of the D ring

1) After water replaces the E ring in the active site (previous slide_.

Figure 11-22

Figure 11-23

E35Q(no acid/base catalysis)NAG2FGLcF

F: magenta atom (stabilizes the carbocation)

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not covered

Figure 11-24

not covered

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The nucleophilic serine can be identified by chemical labeling.

not covered

Box 11-3a

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 11-3c

Box 11-3b

acetylcholine esterase inhibitors

Figure 11-25

Figure 11-26

Figure 11-27

Specificity pockets

Figure 11-28

Not covered

Figure 11-29

X

X

Figure 11-29 part 1

X

Figure 11-29 part 2

X

Figure 11-29 part 3

Figure 11-29 part 4

X

Figure 11-29 part 5

X

Figure 11-30a

Figure 11-30b

Figure 11-31a

BPTI:bovine pancreatic trypsin inhibitor

BPTI is a member of the protein family of Kunitz-type 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 11-31b

Trypsin

BPTI

Figure 11-32

elastase, ph 5.0: elastase, ph 9.0, quick freeze:

Figure 11-32a

Figure 11-32b

Figure 11-33

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.

Box 11-4a

Coagulation Cascade

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

Box 11-4c

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).

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 proton-withdrawing 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

Protease Classes

Serine proteasesThreonine proteasesCysteine proteasesAspartate proteasesGlutamic acid proteasesMetalloproteases