ENZYME BIOCHEMISTRY

Maggy Thenawidjaja Suhartono

Biochemistry of Enzymes
 Biocatalyst
 Involved in biochemical reactions
 Efficient
 Selective
 Protein, except Ribozyme
 Denatured by harsh environtment
 Optimum pH, temperature
 Reduced activation energy
 Cofactor, coenzyme
 Substrate + enzyme = product
 Active site = bind substrate
 Gene product
 How do we name enzyme ?
• Substrate + ase : Lipase Selulase
Protease Pectinase
• Reaction + ase : Dehydrogenase Peroxidase
Oxidase Isomerase
• Trivial : Renin
Subtilisin
• 6 classes of enzymes : EC 1. Oxidoreductase
2. Transferase
3. Hydrolase
4. Lyase
5. Isomerase
6. Ligase
Position : 1, 2, 3
L, D, Cis trans
Chymotrypsin Has A Site for Specificity

O O
N–C–C–N–C–C N–C–C–N–C–C
R H R’
O-
C
Ser
Specificity
Site Catalytic Site

Active Site

Juang RH (2004) BCbasics

 Specificity of Ser-Protease Family
Trypsin Chymotrypsin Elastase
cut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly

O O
O O O O
–C–N–C–C–N–
–C–N–C–C–N– –C–N–C–C–N–
C
C
C CH3
C
Deep and negatively

C
charged pocket

Shallow and
non-polar
NH3 pocket
+

Juang RH (2004) BCbasics

COO- Non-polar
pocket
C
Asp

Active Site
 Stereo Specificity

A
sp3
D
B D C
Enzyme surface C B B
The tetrahedral structure C D
of carbon orbital has rigid
steric strain which makes
the basic building unit of These two triangles are
protein conformation
not identical
Juang RH (2004) BCbasics
 Chiral fine chemicals, $ 16
billion 2007

Enzyme work on organic solvent are usually stereo and regio

selective, they are useful for medical and chemical
industries
TERPENTIN Destilation/fractination Alpha Pinene
Flavour
Enzyme reaction in organic solvent
and
(toluene)
fragrance

Nanik Wijayati, Kusoro Siadi, Hanny Wijaya, MT Suhartono. 2008.

 Substrate Saturation of an Enzyme

A. Low [S] B. 50% [S] or Km C. High, saturating [S]

Experimental procedure for studying Double reciprocal plot for the
the kinetics of the hexokinase hexokinase data. 1/v vs 1/[S] The-y
reaction., v is plotted as afunction of intrecept of 0.01 correspondens to 1/vmax,
[S]. The curve is hyperbolic, so vmax is 100 mol/min the x-intrecept of
approaching vmax -6.7corresponds to -1/km, so km is 0.15
Non competitive inhibition Un competitive inhibition Competitive inhibition
 .
In the presence of the inhibitor,
Km and Vmax become (α/α')Km and (1/α')Vmax,

In these cases, it is usually more practical to treat the

tight-binding inhibitor as an irreversible inhibitor ; however,
it can still be possible to estimate Ki' kinetically if Ki is
measured independently.
Why study Enzyme kinetics?
• Kinetic information is useful for examining possible
mechanisms for the reaction.

• For understanding how metabolism is regulated under

different conditions.

• Useful for understanding pathological states.

• Diseases and disorders often involve alterations in
enzyme activities.

• Understanding how drugs work, because most drugs

function by interacting with enzymes.

• For designing new drugs.

• Many drug molecules are enzyme inhibitors

• Discovery and improvement is an active area of

research in biochemistry and pharmacology.

• A medicinal enzyme inhibitor is judged by its

specificity (its lack of binding to other proteins)
and its potency (its dissociation constant = Ki).

• High specificity and potency ensure that a drug

will have few side effects and low toxicity
 CH2 E
+
CH 2 C O CH2 CH 2

]
N CH 2 Asetil kholin
CH2 CH2
GUGUS SERINE
O

_
+ OH
+ Diisopropilfluorofosfat
H2O
F (DIFP)
Asetil kholin esterase CH3 CH3
CH O P O CH
CH3 CH3
O
-
CH2 COO Asetil

+ kholin
HF
CH2
+ E
HO CH2 CH2 N CH2
CH2
CH2
DIFP : inhibit serine enzyme CH3 O
CH3
CH O P O CH
CH3 CH 3
O
Covalent immobilization of trypsin
on cotton bandage for future use
as an anti-inflammatory agent on
wound dressing.
 Protease inhibitors are important drugs

• Drugs that block or modulate proteases can have dramatic

actions.

• Most natural protease inhibitors are similar to the substrate.

• Several important drugs are protease inhibitors

• Captopril (ACE inhibitor, ACE is metalloproteinase)

• Crixivan (HIV protease inhibitor)
– HIV protease cleaves multidomain viral proteins into their active forms;
blocking this process completely prevents the virus from being infectious.
Replication of HIV Requires an Aspartyl Protease

• Replication of HIV-1 requires proteolytic breakdown of a polyprotein.

Proteolysis is carried out by a virus-encoded protein, HIV protease.

• HIV-1 protease is an aspartyl protease : Asp-Thr-Gly.

• Symmetric dimer with Asp pair at the subunit interface.

• Mutation in Asp : enzyme is inactive.

• Virus needs this protease for multiplication.
• Good target for design of drugs to treat AIDS.
Structure of HIV protease and its binding pocket
B
 • ACE Inhibitor
ACE INHIBITOR

Benazepri)
Captopril
Enalapril
Fosinopril
Lisinopril
Diuretics
\Vitamin
Contain K
 ACE in
several
tissues

Angiotensin-I:
NH2-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe♦His-Leu-COOH
Angiotensin-II:
NH2-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-COOH
 Lineweaver-
BurkInhibitor
Figure 8.1: Lineweaver-Burk plot of initial reaction rates of engineered ACE plot Vand of
engineered
Natural ACE Inhibitor Peptide, showing the relationship to be non-competitive ACE
inhibition
Inhibitor and
Natural ACE
Inhibitor
Peptide,
showing the
relationship to
be non-
competitive
inhibition.
2001 Journal of Biological Chemistry,
276

Bis-indole indirubin active

ingredient of Danggui Longhui
Wan, TCM used in leukemia
described as potent (IC50: 50–100
nm) inhibitors of cyclin-dependent
kinases (CDKs).

indirubins :powerful inhibitors (IC50:

5–50 nm) of an evolutionarily
related kinase, glycogen synthase
kinase-3β (GSK-3β)
GSK-3β, and CDK5 responsible
for most of abnormal
hyperphosphorylation observed in
Alzheimer's

. Indirubins important in treatment of

neurodegenerative disorders
Indirubins inhibit GSK-3β by competing with ATP. Double-reciprocal plots of
kinetic data at different concentrations of indirubin-3′-monoxime.
 Biochem. J. (2010) 425 (149–158

Proliferation of the malaria-

causing Plasmodium falciparum
within erythrocyte is
concomitant with massive
phosphatidylcholine and
phosphatidylethanolamine
biosynthesis. de novo
biosynthesis pathways of both
phospholipids essential for
parasite survival.

PfCK (P. falciparum choline

kinase) and PfEK (P. falciparum
ethanolamine kinase), catalyse
first enzymatic steps of these
essential metabolic pathways.

A inhibition of PfCK with 0, 0.5 & 1mM T3; (B) inhibition of PfEK. the apparent Km (Km′)
plotted as function of inhibitor concentration. Ki values were obtained by intercept with
the x-axis as indicated by the arrow (-Ki)
Enzyme structure and mechanism
 Serine
• Amino acid
• alcohol as residue
 Serine Protease
• Cutting certain peptide
bonds in other proteins

• Activity depends on a set

of amino acid residues in
the active site

• Based on nucleophilic
attack of the targeted
peptidic bond by a serine
 Digestive enzymes

• Chymotrypsin, Trypsin, Elastase

• closely-similar structures
• different substrate specificities

• Pancreatic proteases
 proenzymes trypsinogen and chymotrypsinogen
 synthesized in the pancreas and secreted into
the lumen of the small intestine
Mechanism
Active site
Chymotrypsin Has A Site for Specificity

O O
N–C–C–N–C–C N–C–C–N–C–C
R H R’
O-
C
Ser
Specificity
Site Catalytic Site

Active Site

Juang RH (2004) BCbasics

 Specificity of Ser-Protease Family
Trypsin Chymotrypsin Elastase
cut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, Gly

O O
O O O O
–C–N–C–C–N–
–C–N–C–C–N– –C–N–C–C–N–
C
C
C CH3
C
Deep and negatively

C
charged pocket

Shallow and
non-polar
NH3 pocket
+

Juang RH (2004) BCbasics

COO- Non-polar
pocket
C
Asp

Active Site
 Trypsinogen-Trypsin
• loss of six amino acids from one end
• overall structures remain similar
• Ca++ for thermal stability

activated enzyme
does have more of its
structure organized
into sheets
 Chymotrypsin
• Active site: His57-Ser195-Asp102

• Chymotrypsin cuts on the C-

terminal side of tyrosine,
phenylalanine, and tryptophan
residues
Chymotrypsin
An attack
by OH
Acylation
to C
H transfer
from
Ser to
His
Asp
neutrali
zes the
charge
 Deacylation with Water

Water substitutes
for the amine
component.
-OH attacks the
C=O
Transient
tetrahedral
intermediate
His 57 donates an
H, acid
component is
released
Enzyme is ready for
another
catalysis
Catalytic triad
 Why is chymo specific?
• Where does its specificity for cleaving only phe, trp, and tyr
come from?

• Studies showed that the enzyme has a hydrophobic pocket

called S1 pocket.

• Only phe, trp, and tyr can fit!

• Binding of an appropriate site chains into this pocket positions

the adjacent peptide bond into the active site for cleavage.
Hydrophobic pocket of
Cymotrypsin (S1 pocket)
 Catalytic triads are found in other
hydrolytic enzymes
• Trypsin
• Elastase
• Thrombin
• they all have 40% structural similarity with chymo.
• Operate similar but different substrate specificities
• Trypsin ---> arg and lys
• Elastase---->ala and ser
• Their S pockets are different.
 Cysteine, aspartyl, and metalloproteases are other major
classes of peptide cleaving enzymes

• Not all proteases work on activated serine residues instead

– Cys protease
– Asp protease
– Metalloproteases

• In each of these 3 approaches, a nucleophile is generated

first, it then attacks the peptide carbonyl group.
Chymotrypsin Catalytic Mechanism A1

Catalytic Triad
His57

Asp102 Ser195
H H
O

[HOOC] C N C C [NH2]
N C H C N C
C
O

Check substrate specificity

Chymotrypsin Catalytic Mechanism A2

His57

Asp102 Ser195
H H
O
C C [NH2]
C N
[HOOC] C N C
N C H C
O

First Transition State

 Chymotrypsin Catalytic Mechanism A3

H O
H
C C
C [NH2 ]
C N C N
[HOOC] C
N C H O

Acyl-Enzyme Intermediate
 Chymotrypsin Catalytic Mechanism D1

H O

H O
C C
H
C [NH2 ]
C N C
O

C N-H
[HOOC]
N C H

Acyl-Enzyme Water Intermediate

Chymotrypsin Catalytic Mechanism D2

H
O
C C
O C [NH2 ]
H C N C
O

Second Transition State

Chymotrypsin Catalytic Mechanism D3

H
H O

O C C [NH2]
H C N C
C
O

Deacylation
 Multiple form of enzyme

• Isozyme, different amino acid composition, different MW , can be

separated , similar activities
• As indicator of specific metabolic changes/status

Isozyme ~ Plant Biology

Agronomy, population genetics, Cellular genetics

Systematic, evolution

Isozyme ~ Reliable Single Gene Marker

Identification of cell hybrid
Oligomeric Enzim, > 1 subunit
 > 1 polypeptide chain (subunit)
 High BM
 Most of enzyme : oligomer
 Regulation not through zimogen mechanism, but
through : Feed Back Inhibition
Oligomeric, alosteric
 Lactate dehydrogenase
MW : 4 x 35000 : 140 000, Subunit H & M (amino acid
differences H4, H3M, H2M2 …), Isozyme  different
physiological role)
The presence of specific isozyme is related to deasese
Dimeric enzymes : SS, FS, FF
 DIA Esterase PGM G6PDH

Genetic Diversity in Garlic (Allium sativum L.) as Assessed by AFLPs and

Isozymes
Isoenzyme profiles
 Comparing migration patterns with known standards and controls
 Speciate cell lines.

Aspartate aminotransferase
Glucose-6-phosphate dehydrogenase
Lactate dehydrogenase
Malate dehydrogenase
Time Course of Myocardial Enzyme
Elevations in Serum after Infarction
#3 control specimen method
#8 normal specimen.
# 1 MI patient. LDH and CK elevated Note
# 2 MI patient with previous chest pain
# 4 liver disease
# 5 MI patient; 2 days post MI CK has
almost returned to normal activity and LDH
flip is definite.
# 6 MI patient 1st day post MI; CK elevated
# 7 MI patient complications of heart
failure and passive liver congestion
Change in total superoxide dismutase activity and its isozyme patterns during cotyledon
senescence. DAP = Days after planting
(A)Total SOD activity. Sample weight was 0.5 g per time point
(B) SOD isoforms. 30 μg protein per load . W1, W2, W3 and W4 wild type (WT)
cotyledon at 10, 20, 30 and 40 DAP respectively M1, M2, M3 and M4 mutant type (MT)
cotyledon at 10, 20, 30 and 40 DAP
(C) Different isoforms of SOD using inhibitors. preincubation 5 mM H2O2 or 3 mM KCN.