Download Lecture 12 - Biocatalysis

Catalysis Lecture 12
Biocatalysis
•Introduction to biocatalysis: basic consepts,
terms
•Biotransformations in industry
Enzyme structure
• Enzymes are proteins
• They have a globular
shape
• A complex 3-D
structure
Human pancreatic amylase
© Dr. Anjuman Begum
The active site
© H.PELLETIER, M.R.SAWAYA
ProNuC Database
• One part of an enzyme,
the active site (active
center), is particularly
important
• The shape and the
chemical environment
inside the active site
permits a chemical
reaction to proceed
more easily
Protein chain
Substrate
molecule
Essential groups
outside the
active center
+
-
Catalytic group
Active center
Binding group
The active site
Cofactors
• An additional nonprotein molecule that is
needed by some enzymes
to help the reaction
• Tightly bound cofactors
are called prosthetic
groups
• Cofactors that are bound
and released easily are
called coenzymes
• Many vitamins are
coenzymes
Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file © 2007 Steve Cook
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
The substrate
• The substrate of an enzyme are the
reactants that are activated by the enzyme
• Enzymes are specific to their substrates
• The specificity is determined by the active
site
The Lock and Key Model
• Fit between the substrate and the active site of the enzyme is
exact
• Like a key fits into a lock very precisely
• The key is analogous to the enzyme and the substrate
analogous to the lock.
• Temporary structure called the enzyme-substrate complex
formed
• Products have a different shape from the substrate
• Once formed, they are released from the active site
• Leaving it free to become attached to another substrate
The Lock and Key Model
S
E
E
E
Enzymesubstrate
complex
Enzyme may
be used again
P
P
Reaction coordinate
The Lock and Key Model
• This explains enzyme specificity
• This explains the loss of activity when
enzymes denature
The Induced Fit Model
• Some proteins can change their shape
(conformation)
• When a substrate combines with an enzyme, it
induces a change in the enzyme’s conformation
• Making the chemical environment suitable for the
reaction
• The bonds of the substrate are stretched to make
the reaction easier (lowers activation energy)
• This explains the enzymes that can react with a
range of substrates of similar types
Induced-fit model
The binding induces conformational changes of both
E and S, forcing them to get a perfect match.
Factors affecting Enzymes
•
•
•
•
substrate concentration
pH
temperature
inhibitors
Substrate concentration: Non-enzymic reactions
Reaction
velocity
Substrate concentration
• The increase in velocity is proportional to the
substrate concentration
Substrate concentration: Enzymic reactions
Vmax
Reaction
velocity
Substrate concentration
• Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
• If you alter the concentration of the enzyme then Vmax will
change too.
The effect of pH
Optimum pH values
Enzyme
activity
Trypsin
Pepsin
1
3
5
7
9
11
pH
Pepsin, Trypsin, - protease found in the digestive system , where they
hydrolyses proteins
The effect of pH
• Extreme pH levels will produce denaturation
• The structure of the enzyme is changed
• The active site is distorted and the substrate
molecules will no longer fit in it
• At pH values slightly different from the enzyme’s
optimum value, small changes in the charges of
the enzyme and it’s substrate molecules will occur
• This change in ionisation will affect the binding of
the substrate with the active site.
The effect of temperature
• Q10 (the temperature coefficient) = the increase
in reaction rate with a 10°C rise in temperature.
• For chemical reactions the Q10 = 2 to 3
(the rate of the reaction doubles or triples with
every 10°C rise in temperature)
• Enzyme-controlled reactions follow this rule as
they are chemical reactions
• BUT at high temperatures proteins denature
• The optimum temperature for an enzyme
controlled reaction will be a balance between the
Q10 and denaturation.
The effect of temperature
Q10
Enzyme
activity
0
10
20
30
40
Temperature / °C
Denaturation
50
The effect of temperature
• For most enzymes the optimum temperature is
about 30°C
• Many are a lot lower,
cold water fish will die at 30°C, because their
enzymes denature
• A few bacteria have enzymes that can withstand
very high temperatures up to 100°C
• Most enzymes however are fully denatured at
70°C
Inhibitors
• Inhibitors are chemicals that reduce the rate
of enzymic reactions.
• The are usually specific and they work at
low concentrations.
• They block the enzyme but they do not
usually destroy it.
• Many drugs and poisons are inhibitors of
enzymes in the nervous system.
• They have nothing to do with negative
catalysis
The effect of enzyme inhibition
• Irreversible inhibitors: Combine with the
functional groups of the amino acids in the
active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus, combine
with serine residues in the enzyme
acetylcholine esterase.
Pesticide poisoning
choline esterase
acetylcholine
choline + acetic acid
Acetylcholine accumulation will cause
excitement of the parasympathetic system:
vomitting, sweating, muscle trembling, pupil
contraction
+ E OH
P
R'O
RO
X
RO
+
P
O
organophosphate
O E
R'O
AChE
O
acid
inhibited AChE
+
N
HX
CHNOH
CH3
E OH
PAM
O
OR'
P
+
N
CH3
CHNO
OR
Heavy metal poisoning
• Heavy metal containing chemicals bind to the –SH
groups to inactivate the enzymes.
S
SH
+
E
Hg2+
SH
2H+
S
Cl
+
E
+
S
SH
SH
Hg
E
Cl
As C
H
CHCl
E
S
As C
H
CHCl
+
2HCl
The effect of enzyme inhibition
• Reversible inhibitors: These can be
washed out of the solution of enzyme by
dialysis.
There are two categories.
The effect of enzyme inhibition
1. Competitive: These
compete with the
substrate molecules for
the active site.
The inhibitor’s action is
proportional to its
concentration.
Resembles the substrate’s
structure closely.
E+I
Reversible
reaction
EI
Enzyme inhibitor
complex
The effect of enzyme inhibition
Fumarate + 2H++ 2e-
Succinate
Succinate dehydrogenase
CH2COOH
COOH
CHCOOH
CH2
CH2COOH
COOH
Malonate
CHCOOH
The effect of enzyme inhibition
2. Non-competitive: These are not influenced by
the concentration of the substrate. It inhibits by
binding irreversibly to the enzyme but not at the
active site.
Examples
• Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
• Heavy metals, Ag or Hg, combine with –SH
groups.
These can be removed by using a chelating agent
such as EDTA.
Applications of inhibitors
• Negative feedback: end point or end product
inhibition
• Poisons snake bite, plant alkaloids and nerve
gases.
• Medicine antibiotics, sulphonamides,
sedatives and stimulants
Enzymes – key elements for biocatalysis
Enzyme class
Catalyzed reaction
Oxidoreductases
Oxidation-reduction reaction
Transferases
Transfer of functional group
Hydrolases
Hydrolytic reactions
Lyases
Group elimination (forming double
bonds)
Isomerizaion reaction
Isomerases
Ligases
Bond formation coupled with a
triphosphate cleavage
Accelerated reaction rates
Non-enzymatic
rate constant
(kn in s-1)
Enzymatic
rate constant
(kn in s-1)
10-1
106
Chymotrypsin
4 x 10-9
4 x 10-2
Lysozyme
3 x 10-9
5 x 10-1
Triose phosphate
isomerase
4 x 10-6
4 x 103
Urease
3 x 10-10
3 x 104
Mandelate racemase
3 x 10-10
5 x 102
Alkaline phosphatase
10-15
102
Enzyme
Carbonic anhydrase
Kinetics
Reaction rate
[P]
[P]
Initial slope = vo =
t
0
Time (t)
[P]
t
Initial velocity
• The reaction rate is defined as the product
formation per unit time.
• The slope of product concentration ([P])
against the time in a graphic representation
is called initial velocity.
• It is of rectangular hyperbolic shape.
Reaction velocity curve
V0
Vmax
Vmax/2
[S]
0
Km
Intermediate state
Forming an enzyme-substrate complex, a
transition state, is a key step in the catalytic
reaction.
k1
E + S
ES
k3
E + P
k2
initial
intermediate
final
Free energy
transition state, S
G+ (uncatalyzed)
G+ (catalyzed)
reactants
G for
the reaction
products
Reaction progress
Rate constants
k1
E + S
ES
k3
E + P
k2
• k1 = rate constant for ES formation
• k2 = rate constant for ES dissociation
• k3 = rate constant for the product released
from the active site
Michaelis-Menten Equation
• The mathematical expression of the
product formation with respect to the
experimental parameters
• Michaelis-Menten equation describes the
relationship between the reaction rate and
substrate concentration [S].
Assumptions
• [S] >> [E], changes of [S] is negligible.
• k2 is negligible compared with k1.
• Steady-state: the rate of E-S complex
formation is equal to the rate of its
disassociation (backward E + S and forward
to E + P)
[S]
V = K
V
max
m + [S]
Describing a hyperbolic curve.
Km is a characteristic constant of E
[S] << Km ，v ∝ [S]
[S] >> Km ，v ≈ Vmax
V0
Vmax
Zero order with
respect to [S]
First order with
respect to [S]
0
[S]
Significance of Km
• the substrate concentration at which
enzyme-catalyzed reaction proceeds at onehalf of its maximum velocity
• Km is independent of [E]. It is determined
by the structure of E, the substrate and
environmental conditions (pH, T, ionic
strength, …)
V0
Vmax
Vmax/2
[S]
0
Km
• Km is a characteristic constant of E.
• The value of Km quantifies the affinity of the
enzyme and the substrate under the
condition of k3 << k2. The larger the Km，
the smaller the affinity.
k2 + k3
Km =
k1
Km for selected enzymes
Enzyme
Substrate
km
Catalase
H2O2
25
Hexokinase
ATP
0.4
D-Glucose
0.05
D-Fructose
1.5
Carbonic anhydrase
HCO3-
Chemotrypsin
Glycyltyrosinylglycine
108
N-Benzoyltyrosinamide
2.5
9
Galactosidase
D-Lactose
4
Threonine dehydratase
L-Threonine
5
Significance of Vmax
• The reaction velocity of an enzymatic
reaction when the binding sites of E are
saturated with substrates.
• It is proportional to [E].
Turnover number
k3 = Vmax / [E]
• Vmax is the reaction rate when the enzymes
are saturated, and is independent of the
enzyme concentration.
• The number of the products converted in a
unit time by one enzyme molecule which is
saturated.
Lineweaver-Burk plot
Km 1
1
1
+
=
Vmax [S]
Vmax
V
• To determine Km and Vmax
• To identify the reversible repression
Double-reciprocal plot
1/V
Slope = Km/Vmax
Intercept = -1/ Km
Intercept = 1/Vmax
1/[S]
Enzyme activity
• Determination of the enzymatic activity
requires proper treatment of enzymes,
excess amount of substrate, optimal T and
pH, …
• One katal is the amount of enzyme that
converts 1 mol of substrate per second.
• IU = 16.67×10-9 kat
Fermentation and Biotransformation
Enzymatic processes
Examples
Fermentation of glucose to alcohol
Yeast metabolize glucose for their metabolic
processes
Anaerobic glycolysis – Production of 2
equivalents of ATP for cells energy per
equivalent of glucose metabolized
While this process is underway, two
enzymatic pathways are coupled that
use/regenerate NAD – the cofactor in the
enzyme – alcohol dehydrogenase
O
O
O
O
NADH + H+
NAD+
+
O
CH3
pyruvate
HC
H2C
O
CH3
acetaldehyde
alcohol
dehydrogenase
CH3
ethanol
OH
Chemistry of Alcohol Dehydrogenase
A. Reduction of acetaldehyde
•
Normally in the metabolic process of yeast, acetaldehyde is reduced to ethanol
by the addition of hydrogen across the carbon-oxygen double bond by the
enzyme
NADH + H+
NAD+
H
H
H3C
H3C
C
O
acetaldehyde
•
alchohol
dehydrogenase
C
H
O
H
ethanol
This reaction is very similar to the sodium borohydride reduction of
aldehydes and ketones
H
H
H3C
O
NaBH4
R-OH
H3C
H
O
H
acetaldehyde
ethanol
B. Enzymatic Reduction
1. Hydride source
Within the enzyme, the hydride source is nicotinamide dinucleotide
(NAD/H):
O
H
O
H
H
C
C
NH2
NH2
O
-
O
P
O
N
O
-
O
H
H
OH
H
H
O
H
P
O
N
O
H
H
OH
H
H
+ H
O
H
H
OH
H
H
reduced nicotinamide dinucleotide, NADH
H
P
N
ON
O
hydride
NH2
N
O
N
N
O
O
N
-
O
P
H
NH2
N
O
O
N
O
H
H
OH
H
H
H
nicotinamide dinucleotide, NAD+
In biological systems, a reagent such as NaBH4 (chemical reduction)
is too strong, and the donation of hydride is NOT reversible
Once more NaBH4 reacts with water to produce hydrogen gas so it cannot
exist long or be regenerated in a living system
From NADH to NAD+, the donation of hydride is an equilibrium process
(controlled by Le Chatlier’s principle), as both the oxidized (NAD+) and
reduced (NADH) forms are roughly equal in energy
The equilibrium constant for this process under biological conditions is
close to one.
The reasons for come from the stability of both forms of NAD:
H
O
H
O
H
C
C
NH2
- H
NH2
N
N
R
R
+ H
hydride
NAD+
NADH
+ H
H
Large concentration
of positive charge
density in the
contributing resonance
structure favors reverse
reaction.
O
C
NH2
N
R
Contributing resonance structure
Hydride donation
leads to formation
of an aromatic ring,
Favoring the forward
reaction
Mechanism of reduction (acetaldehyde)
H
O
H3C
Base
O
Base
H
H3C
H
H
H
O
H
O
H
H
C
C
NH2
NH2
N
N
R
R
NADH
NAD+
Of course in biological systems, the reverse reaction also occurs:
O
Base
H
H3C
H
O
H3C
H
H
H
O
O
H
H
H
C
C
NH2
NH2
N
N
R
R
NAD+
NADH
In fact, the enzyme itself is named for this reverse reaction:
alcohol dehydrogenase
Base
2. Active site of the Enzyme:
Unlike the NaBH4 reduction, this reaction is stereospecific
Active site of enzyme must provide three point recognition of the
substrate
Active site must also provide the the second hydrogen, as H+ to
complete the reduction of the C=O in the forward reaction and accept
H+ in the reverse reaction
The active site of alcohol dehydrogenase
Histidine 67
H
N
Acetaldehyde
Enters from the
bottom
N
Cysteine 48
H2
C
Zn+2
S
S
H2
C
Cysteine 174
O
H
O
H2
C
Serine 48
O
H3C
H3C
C
H
C
Recognition occurs
through the Zn+ atom,
the protonated base,
and NADH
H
O
H
H
C
NH2
N
O
R
H3C
C
NADH
H
H
H
Hydride is added from
Upper face of acetaldehyde
Antibiotics
Penicillin and 6-Aminopenicillanic Acid (6-APA)
Penicillin:
First discovered by Fleming in 1932
19% of worldwide antibiotic market.
Superior inhibitory action on bacterial cell wall synthesis
Broad spectrum of antibacterial activity
Low toxicity
Outstanding efficacy against various bacterial strains
Excessive use has led to development of resistant pathogens
6-APA: Raw material for production of new semisynthetic penicillins
(amoxycillin and ampicillin)
Fewer side effects
Diminished toxicity
Greater selectivity against pathogens
Broader antimicrobial range
Improved pharmacological properties
Chemical and Enzymatic Deacylation
of Penicillins to 6-APA
R
C
H
N
S
O
N
CH3
CH3
Penicillin acylase
COOH
O
Penicillin V or G
[R=Ph or PhO]
S
NH2
Alkaline
[Enzymatic]
N
O
(6-APA)
R
PCl5
ROH
H2O
[Chemical]
Pyridine
Me3SiCl
C
H
N
S
O
N
O
CH3
CH3
COOSiMe3
CH3
CH3
COOH
Penicillin and 6-Aminopenicillanic Acid (6-APA)
Chemical method:
Use of hazardous chemicals - pyridine, phosphorous
pentachloride, nitrosyl chloride
Enzymatic method:
Regio- and stereo-specific
Mild reaction conditions (pH 7.5, 37 oC)
Enzymatatic process is cheaper by 10%
Enzymes:
Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium,
Streptomyces lavendulae
Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium,
Fusarium sp., Pseudomonas cidovorans
Immobilized Enzyme:
Life, 500-2880 hours
Aspartame (L-Asp-L-Phe-Methyl Ester)
Aspartame is dipeptide sweetener formed by linking the methyl ester
of phenylalanine with aspartic acid
Extensively used in food and beverages
200 times as sweet as sucrose
Annual sale: 200 million kg, $ 850 million
Nutrasweet Corp. retains 75% of the US market
Chemical method:
The amino group of aspartic acid needs to be protected to prevent its reacting
with another molecule of aspartic acid to give unwanted by-products
The correct single enantiomer of each of the reactants must be used to give
the required stereochemistry of aspartame (beta-aspartame is bitter tasting)
Enzymatic method:
Thermolysin promotes reaction only at the alpha-functionality
Mild condition, pH 6-8, 40 oC
Biocatalytic Production of Aspartame
HO2C
Ph
+
PhCH2OCNH
CO2H
O
N-Cbz-aspartic acid
H2N
CO2Me
D,L-phenylalanine
Methyl ester
thermolysin
H2O
HO2C
PhCH2OCNH
Cbz, benzyloxycarbonyl
O
Ph
CNH CO2Me
O
Cbz-aspartame
Important Factors in Using Enzymes
• Reactions possible that are not possible using chemistry
• Specificity of reaction including substrate specificity, positional
specificity, stereospecificity
• Allows milder process conditions e.g. temperature, pH, sterility etc.
• Reduces number of process steps required
• Eliminates the need to use organic solvents in processing
• Immobilization of enzyme to allow its reuse or continuous use
• Use of enzymes in combination with other separate chemical steps
• Genetic engineering to improve enzymes
Environmentally Compatible Synthesis of
Catechol from Glucose
acetone
a
hydroquinone
b
HO
benzene
cumene
CO2H
OH
O
HO
CO2H
OH
d
OH
OH
D-glucose
(a)
(b)
(c)
(d)
phenol
OH
HO
d
d
O
c
OH
catechol
HO
OH
OH
3-dehydroshikimic protocatechuic
acid
acid
propylene, solid H3PO4 catalyst, 200-260°C, 400-600 psi.
O2, 80-130°C then SO2, 60-100°C.
70% H2O2, EDTA, Fe2+ or Co2, 70-80°C.
E. coli AB2834/pKD136/pKD9.069A, 37°C.
Draths and Frost, 1995
Biological function of some metals
Na, K Charge transfer, ostmotic equilibrium
Mg
Structure, Hydrolase, Isomerase
Ca
Structure, charge transfer, releaser
V
Oxidase, N2 binding
Cr
Unknown – glucose tolerance
Mo
Oxidase, nitrogen binding, oxo gruop transfer
W
Dehydrogenase
Mn
Photosynthesis, oxidase, structure
Fe
Oxidase, O2 transport and storing, electron transfer, N2 binding
Co
Oxidase, alkyl group transfer
Ni
Hydrogenase, hydrolase
Cu
Oxidase, O2 transport, electron transfer
Zn
Structure, hydrolase
Catalysis by Carbonic anhydrase
(carbonate dehydratase)
H
O_
H+
H
Zn
His94
His119
His96
H
CO2
O
His94
His96
O
Zn
His119
C
H
O_
HCO3-
O
Zn
His94
His119
His96
H2O
H
O
O
O
_
O
Zn
His94
His119
His96
H
_
O
O
Zn
His94
His119
His96
Industrial Enzyme Market
Annual Sales: $ 1.6 billion
Food and starch processing:
Detergents:
Textiles:
Leather:
Pulp and paper:
45%
34%
11%
3%
1.2%
Biotransformations in industy
References
• P. Billiet „Enzymes”
• http://202.118.40.5/biochemistry/ewebeditor/uploadfile/20091117093812
957.ppt