Biochemistry - Study Guide - Chapters 9-10

Major
1.
2.
3.

classes of enzymes
Oxidoreductases oxidation-reduction rxns
Transferases group transfer e.g., NMP kinase
Hydrolases hydrolysis rxns (transfer of fxnal groups to water) e.g.,
chymotrypsin
4. Lyases addition or removal of groups to form double bonds
5. Isomerases isomerization (intramolecular group transfer)
6. Ligases ligation of two substrates at expense of ATP hydrolysis

Catalytic Strategies
1. Covalent catalysis
Enzyme and substrate become linked in a covalent bond at step(s) in rxn
pathway
Formation of covalent bond provides chemistry that speeds rxn
Covalent bond is transient: later bond is broken so enzyme returns to free
state if it were permanent it would inhibit enzymatic activity
e.g., serine proteases
2. General acid-base catalysis
Catalysis in which a proton is transferred in the transition state
Specific acid-base catalysis involves H or OH that diffuses into catalytic
center
General acid-base catalysis involves acids and bases other than H and OH
that facilitate the transfer of H in the transition state
3. Catalysis by approximation (not covered)
4. Metal ion catalysis
Metal ion (Zn) facilitates formation of nucleophiles such as OH
e.g., catalysis by carbonic anhydrase
Serine proteases
Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin
All involve serine in catalysis
Serine is always in active site as part of catalytic triad (Ser-His-Asp)
Homologous, but locations of catalytic triad residues may differ
Chymotrypsin
cleaves peptide bond near aromatic (Phe) or large hydrophobic side chains (Met)
inactivated by tx with DIPF, a group-specific reagent that reacts only with Ser195
in active site
chromogenic substrate: cleavage results in colored pdt
chymotrypsin hydrolyzes ester bond: pdt P-Nitrophenolate
can monitor enzymatic activity based on color observed
2 products appear with different kinetics: burst of P-Nitrophenolate, lag
of acetate
bc hydrolysis by chymotrypsin takes place in two stages: acylation to form
acyl-enzyme intermediate (and release of 1st pdt) followed by deacylation to
regenerate the free enzyme (and release of 2 nd pdt)
synthesized as single peptide, cleaved into 3 chains, folded with side chains of
catalytic residues lining the active site in the upper half of the structure
catalytic triad: Asp102-His57-Ser195: His acts as general base by accepting H from
Ser when substrate is bound (Ser OH is nucleophile but is a better nucleophile as
O- alkoxide ion)

Peptide hydrolysis by Chymotrypsin:

1.
2.
3.
4.
5.
6.
7.
8.

Substrate binds
His takes H from Ser-OH, forms oxyanion hole - nucleophilic attack of Ser on carbonyl
Collapse of tetrahedral intermediate
Release of amine pdt
Water binds will serve as nucleophile
Nucleophilic attack of water on acyl-enzyme intermediate
Collapse of tetrahedral intermediate
Release of carboxylic acid pdt

Oxyanion hole structure stabilizes tetrahedral intermediate via interactions with

protein NH grps
Pocket specificity chymotrypsin specifies the S1 pocket, a deep & relatively
hydrophobic pocket, where only long uncharged side chains of residues such as
Phe and Trp can fit; also Ser-195 is positioned to cleave peptide backbone
between residue bound in pocket and next residue in sequence

Specificity of serine proteases for different substrates

Potential sites of interaction of
substrate with enzyme are
designated P and corresponding
binding sites on enzyme are
designated S. Carboxyl terminal
side of scissile bond designated
with

Scissile
bond

All serine proteases have catalytic

triad specificity is due to S1
pocket. (Trypsin & Elastase
homologs of Chymotrypsin)
Trypsin has (-) Asp at bottom of
pocket prefers interaction with
(+) Arg or Lys. Elastase has shallow
pocket due to Val bulky side chains
prefers small side chains.

Carboxypeptidase II
Catalytic triad composed of same residues as chymotrypsin but lacks structural
similarity
Site-directed mutagenesis of subtilisin
Mutations in any component of catalytic triad cause dramatic loss of enzymatic
activity
Determine relative enzymatic activities by altering 1 residue at a time
Conversion of active-site Ser or His to Ala dramatically reduced catalytic power: k cat
value fell to less than one-millionth of its value for wild-type enzyme
Conversion of Asp reduced catalytic power by less, but still a significant decrease
Simultaneous conversion was no more deleterious than Ser or His alone the
catalytic triad, and specifically the Ser-His interaction, act together to generate a
nucleophile of sufficient power to attack the carbonyl carbon of peptide bond
Cysteine proteases
Similar to chymotrypsin family serine proteases except that a Cys is activated by
His to attack peptide
e.g., papain enzyme
Aspartyl proteases
All involve 2 Asp residues at active site that work together as general acid-base
catalysts with water as nucleophile
1 Asp has lower pKa, 1 Asp has higher pKa deprotonated Asp acts as general
base, accepting H from water, forming OH in transition state. Other Asp acts as
general acid, donates H to C=O, formation of tetrahedral intermediate.
Most have 2 lobed symmetrical tertiary structure
e.g., pepsin, chymosin, cathepsin D, renin, HIV-1 protease
HIV-1 Protease
cleaves the polyprotein products of HIV genome
imitation of aspartic proteases (two Asp residues of differing pK as)
is homodimer of identical subunits (4 structure) more efficient use of
genes
has flaps that close over binding pocket after substrate binds
required to cleave nonfxnal polyprotein into smaller fxnal proteins
protease inhibitors (e.g., Idinivar) used as HIV drug tx (must be able to kill
virus w/o blocking other essential proteases in body)
Metalloproteases

active site contains bound metal ion (almost always Zn) that activates water
molecule to act as nucleophile to attack peptide bond
can bind multiple ligands, Zn has 4 coordinates
e.g., Thermolysin

Carbonic Anhydrase rxn

CO2 in gas state must dissolve in water
Hydration enzyme: carbonic anhydrase
Pdt is carbonic acid which is deprotonated to form bicarbonate ion (HCO 3-)
Structure: Zn with four coordinates, 3 His and 1 water (serves as nucleophile)
Effect of pH on carbonic anyhdrase activity: increasing pH = increased activity
(max at pH 8)
pKa of water = 15.7, but pKa of Zn-bound water = 7 bc Zn has (+) charge and can
draw proton to help dissociation
Mechanism: Zn-bound water gives up H, OH can now attack C=O, displacement of
bicarbonate ion
Synthetic analog model
Can synthesize analogous Zn bound compound when bound to water has pK a 8.7
(vs 7 in carbonic anhydrase)
At pH 9.2: complex accelerates hydration of CO 2 >100 fold
Kinetics of water deprotonation
For Zn-bound H2O dissociation of H: dissociation constant k = 10 -7 s-1
Need H to diffuse away: k 104 s-1
But if you have buffer it can accept H (must faster than diffusion): k 106 s-1
Increase buffer concentration = increase enzymatic activity of carbon dioxide
hydration
Buffer cant get in active site His on outside surface takes up H, then buffer can
accept H from His: proton hopping or proton transfer
Enzymatic control and regulation
1. Allosteric control: positive and negative effectors
2. Covalent modification: add/remove phosphate via protein phosphatase
3. Proenzyme (zymogen) Enzyme conversion: activation by proteolysis (enzyme
cleavage) & conformational change
4. Multiple forms of enzymes (isoforms): diff sequence but catalyze same rxn
5. Amount of enzymes: related to transcriptional control
1. Allosteric enzymes
An oligomer whose biological activity is affected by other substances binding to
it that change the enzymes activity by altering the conformation of 4 structure
Allosteric effector: substance that modifies behavior of allosteric enzyme
Allosteric inhibitor
Allosteric activator
Allosteric regulation: enzymes situated at key steps in metabolic pathways are
modulated by allosteric effectors usually produced elsewhere in pathway
Allosteric kinetics are sigmoidal (not M-M hyperbolic shape)
May be feedforward activator or feedback inhibitor (end pt is allosteric enzyme
so when you build up pdt it will inhibit its own production)

e.g., pyrimidine biosynthesis via ATCase (aspartate transcarbamoylase): end pdt is

CTP which feedback-inhibits enzymatic activity of ATCase
ATCase: 2 types of subunits: 3 regulatory dimers and 2 catalytic trimers each
regulatory dimer interacts with catalytic trimer via Zn domain
Zn domain: Zn ion bound to four Cys residues
PALA: bisubstrate analog that resembles an intermediate along catalytic pathway
(can bind and stabilize structure to enable identification of 3D structure
otherwise rxn goes too fast to see)
ATCase active site: composed mainly of residues from one subunit but an adj
subunit also contributes important residues Lys and Ser
T-state (no substrate) R-state via bisubstrate analog PALA: two catalytic trimers
separate by 12 and rotate 10, regulatory dimers rotate 15
CTP binds to regulatory subunits and stabilizes T-state, decreasing enzymatic
activity
R-T Equilibrium (L)
L = [T] / [R]
R-state curve: max activity; T-state curve: min activity
Sigmoidal curve in between these: actual observed activity
Larger L value = more T = less activity
Smaller L value = more R = more activity
Allosteric effectors
Inhibition (CTP): stabilize T-state, increases L
Activation (ATP): stabilize R-state, decreases L
Effect of positive modulators on R-T Equilibrium (L)
Homotropic effect: substrate binding to active site increases binding affinity
of other substrate
Heterotropic effect: non-substrate effectors' binding to regulatory domain
either activates or inhibits
2. Covalent Modification
May inhibit or activate depending on enzyme
Phosphorylation by kinases
Occurs on 3 residues: Ser, Thr, Tyr (all contain OH)
Two different kinases: 1 phosphorylates Ser/Thr; other phosphorylates Tyr
ATP common donor of P: protein kinase cleaves -phosphate on ATP ADP,
transfers -P to residue (Ser, Thr, Tyr)
Many signal transduction pathways involve kinase activity
Activation of Protein Kinase A (PKA) by cAMP
o PKA has two regulatory subunits, 2 catalytic subunits (in absence of
cAMP, R2C2 complex is inactive)
o PKA regulatory domain: bind cAMP and contains pseudosubstrate
sequence with Ala (vs. Ser/Thr in consensus sequence)
o Binding of pseudosubstrate to PKA via charge-charge & hydrophobic
interactions
o Binding of four molecules of cAMP to R domain initiates
conformational change & release of pseudosubstrate sequence:
activates PKA by dissociating regulatory domains from catalytic active
domains
o So binding of cAMP to regulatory subunit relieves its inhibition of the
catalytic subunit

De-phosphorylation by phosphatases
Remove P to return protein to original form
Phosphorylation/Dephosphorylation
NOT the reverse of each other
Each rxn is essentially irreversible
Each rxn utilizes different enzyme (kinase vs phosphatase)
Both rxns take place at negligible rates w/o enzymes
Process is reversible, but single rxns are irreversible

3. Activation of pro-proteins by proteolytic cleavage

Conversion of zymogens (pro-enzymes) to active enzymes
e.g., pepsinogen pepsin
e.g., trypsinogen trypsin
e.g., chymotrypsinogen chymotrypsin
Secretion of zymogens by acinar cell of pancreas
Zymogen proenzymes synthesized in acinar cells of pancreas
Stored inside membrane-bound granules
Waits for signal to release/secrete

Proteolytic activation of chymotrypsin

Chymotrypsinogen requires activation by trypsin -Chymotrypsin
-Chymotrypsin is active on other -Chymotrypsin to remove two dipeptides
(14/15 and 147/148 in sequence) -Chymotrypsin
-Chymotrypsin has 3 peptide chains linked by 2 disulfide bonds
cleavage at 15 creates new N-terminal at Ile-16 which moves inward to
interact (charge-charge interaction) with Asp at active site
Proteolytic activation of trypsin
Enteropeptidase hydrolyzes unique Lys-Ile peptide bond in trypsinogen
trypsin
Trypsin is common activator of all pancreatic zymogens (e.g.,
chymotrypsinogen chymotrypsin)
Interaction of trypsin with its inhibitor (pancreatic trypsin inhibitor): bound
inhibitor and free inhibitor has same structure no need for conformational
change inhibitor is very effective substrate analog but binds to active site
very tightly and is turned over slowly

4. Regulation of Iso-Enzymes
Similar but not identical sequences
Catalyze same rxn but may have different kinetics
May have tissue specification
Display different kinetic parameters (Km) or respond to different regulatory
molecules
Existence of isozmes permits fine-tuning of metabolism to meet needs of given
tissue or developmental stage

e.g., isozymes of Lacate Dehydrogenase (LDH) vary by tissue (mostly heart vs

liver)