Download Enzymes

Enzymes
• Catalysts speed up chemical
reactions without being
changed themselves
• Living organisms make
biological catalysts called
enzymes
• Enzymes are globular proteins
which act as catalysts of
chemical reactions
• Without enzymes to catalyze
them, many chemical
processes happen at a very
slow rate in living organisms
• By making some enzymes and
not others, cells can control
what chemical reactions
happen in their cytoplasm
Introducing
Enzymes
• The structure of enzymes
is quite delicate and can
be damaged by various
substances and
conditions
• This is called denaturation
– Changing the structure of
an enzyme (or other
protein) so that it can no
longer carry out is function
• Denaturation is usually
permanent in chemical
reactions one or more
reactants are converted
into one or more products
Introducing
Enzymes
Introducing
Enzymes
• In reactions catalyzed by enzymes, the reactants are
called substrates
substrate(s)
sucrose + H2O
enzyme
sucrase
product(s)
glucose +
fructose
Enzyme-Substrate Specificity
• Most enzymes are specific
– They catalyze very few
different reactions
• They therefore only have a
very small number of
possible substrates
– this is called enzyme-substrate
specificity
• The substrates bind to a
special region on the
surface of the enzyme called
the active site
• An active site is a region on
the surface of an enzyme to
which substrates bind and
which catalyze a chemical
reaction involving the
substrates
Enzyme-Substrate Specificity
• The active site of an enzyme has
a very intricate and precise shape
• It also has distinctive chemical
properties
• Active sties match the shape and
chemical properties of their
substrates
• Molecules of substrate fit the
active site and are chemically
attracted to it
• Other molecules either do not fit
or are not chemically attracted
– They do not therefore bind to the
active site
• This is how enzymes are
substrate-specific
• The way in which the enzyme
and substrate fit together is
similar to the way in which a key
fits a lock
• The enzyme is like the lock and
the substrate is the key that fits it
Enzyme induced fit
substrate
enzyme
• When the substrate enters the active site, the
enzyme conforms (changes shape) and “hugs”
the substrate
•
•
•
•
•
•
•
Substrate molecules
are in continual
random motion
If one collides with
the active site, it can
bind to it
The substrate fits the
active site
If other molecules
collide with the active
site they do not fit
and fail to bind
The active site
catalyzes a chemical
reaction
The substrates are
turned into products
The products detach
from the active site,
leaving it free for
more substrate to
bind
Stages in Enzyme Catalysis
rate of reaction
At low substrate concentrations, the enzyme
activity is directly proportional to substrate
concentration. This is because random collisions
between substrate and active site happen more
frequently with higher substrate concentrations
At high substrate
concentrations, all the
active sites of the
enzyme are fully
occupied, so raising the
substrate concentration
has no effect
concentration of substrate
Effect of Substrate Concentration on
Enzyme Activity
rate of reaction
how enzyme concentration
affects rate of reaction
enzyme concentration
Factors Affecting Enzyme Activity
• Wherever enzymes are used, it is
important that they have the conditions
that they need to work effectively
• Temperature, pH, and substrate
concentration all affect the rate at which
enzymes catalyze chemical reactions
• Enzymes, unlike inorganic catalysts, have
optimum conditions under which they
work.
Effect of Temperature
• Enzyme activity increases as
temp increases, often
doubling with every 10 oC rise
• This is because collisions
between substrate and active
site happen more frequently
at higher temps due to faster
molecular motion
• At high temperatures
enzymes are denatured and
stop working
• This is because heat causes
vibrations inside enzymes
which break bonds needed to
maintain the structure of the
enzyme
Effect of pH
• Every enzyme
has very narrow
range of pH
within which it
works properly.
• Increasing or
decreasing pH
– denatures the
protein &
slows/stops
reaction rate
• different enzymes
have different
optimal conditions
for working properly
Ex:
temperature
pH level
Using Enzymes in Biotechnology
• Biotechnology is the use or organisms or
parts of organisms to produce things or to
carry out useful processes
• There are many ways in which enzymes,
obtained from living organisms, can be
used in biotechnology
The Use of Pectinase in Fruit Juice Production
• Pectin is a complex polysaccharide, found in cell walls of plants
• Pectinase is an enzyme that breaks down pectin by hydrolysis reactions
• Source of enzyme:
– Pectinase is obtained by artificially culturing a fungus (Aspergillus niger)
– The fungus grows naturally on fruits, where it uses pectinase to soften the cell
walls of the fruit so that is can grow through it
• Use of pectinase in biotechnology:
– Fruit juices are produced by crushing ripe fruits to separate liquid juice from solid
pulp
– When ripe fruits are crushed, pectin form links between the cell wall and the
cytoplasm of the fruit cells, making the juice viscous and more difficult to
separate from the pulp
– Pectinase is added during crushing of fruit to break down the pectin
• Advantages:
– Pectinase makes juice more fluid and easy to separate from the pulp
– It therefore increases the volume of juice that is obtained
– It also makes the juice less cloudy by helping solids suspended in the juice to
settle be separated from the fluid
Use of Protease in Biological Washing Powder
• Protease enzymes break down proteins into soluble particles and amino
acids
• Laundry washing powders that contain protease are called biological
washing powders
• Source of the enzyme:
– Protease is obtained by culturing a bacterium, Bacillus licheniformis, that is
adapted to grow in alkaline conditions
– This bacterium feeds on proteins in its habitat by secreting protease
– The protease has a high pH optimum between 9 and 10
• Use of protease in biotechnology:
– Detergents in laundry washing powders remove fats and oils during the washing
of clothes, but much of the dirt on clothing is made of protein, not lipids
– If proteases added to the washing powder, this protein is digested during the
wash
– The high pH optimum of the protease allows it to remain active, despite the high
pH caused by alkalis in the washing powder
• Advantages:
– If protease is not used, protein stains on clothes can only be removed by using a
very high temperature wash
– Protease allows much lower temperatures to be used, with lower energy use and
less risk of shrinkage of garments or loss of colored dyes
Types of Enzymes
•
Transferases:
–
•
Hydrolases:
–
•
Enzymes that catalyze the removal of a groups from substrates by
mechanisms other than hydrolysis
Isomerases:
–
•
enzymes that catalyze the hydrolysis of esters, cabs and proteins
Lyases:
–
•
Enzymes that catalyze the transfer of a functional group between
two substrates
Enzymes that catalyze the interconversion of stereoisomers and
structural isomers.
Ligases:
–
Enzymes that catalyze the linking of two compounds by breaking a
phospate anhydride bond in ATP
bread and cheese making
detergents
biosensor
medicines
Industrial Uses of Enzymes
•
•
•
•
•
Lactase used to convert lactose into galactose
for lactose intolerant
Amylase, glucose isomerase and glucoamylase
are used to convert starch into high fructose
syrup
Pectinase replaces harsh toxic chemicals in
preparing cotton for dyeing
Cellulase used to “stonewash” denim and to
break down wood chips into paper pulp
Medical uses: proteases called plaminogen
activators used to break down blood clots
Control of enzyme function:
I.
Inhibition
prevent the substrate from
binding the active site
a. competitive inhibitor
inhibitor molecule
directly binds the active
site and blocks substrate
b. noncompetitive inhibitor
does not bind the active
site directly, but causes a
change in conformation
(shape) in the enzyme –
the shape of the active
site is changed
Enzyme cofactors
• these non-protein factors can be
organic molecules (coenzymes) or
inorganic ions ( Ca+2 or Zn- )
• they enhance the enzyme’s activity
enzyme
+
coenzyme
Irreversible inhibitors:
poisons
substrate
active site
Heavy metals often
bond with a thiol
group (S-H) present
on the cysteine amino
acid.
Hg
enzyme
They can also bind
ionically with COOH,
and OH groups on
side chains of amino
acid residues.
mercury binds the active site permanently and
competitively inhibits the papain enzyme
Mercury Poisoning
•
Hg is believed to bond with SAM** (S-Adenosyl
methionine) making it inactive
•
SAM is a coenzyme used in transferring methyl groups
to proteins, nucleic acids and lipids
•
Used in about 40 metabolic pathways
**actual mechanism is unknown, but Hg causes damage to central
nervous system, endocrine system and kidneys and other organs
Mercury Poisoning
Elemental Mercury
–
Vapor is bad, liquid is not readily absorbed
–
Vapor present in fluorescent lights,
Mercury Salts
–
–
Dissolve in water, absorbed by gastrointestinal tract
•
Can't break blood-brain barrier
•
Cause kidney damage but not neurological damage.
Used in industry, coal burning mostly
•
Coal fired plants are largest source (mercury video)
•
Also comes from incineration of waste
•
(can be used in manufacture of high fructose corn syrup, but new technologies in
the US are Hg free...hopefully our HFCS is manufactured in the US)
Organic mercury
–
Highly toxic: dimethylmercury, few μL = death
–
Hg salts are put into water by industry where it reacts with organic substances
and accumulates in biological species: fish, particularly tuna, shark, swordfish
Lead Poisoning
•
Lead replaces the zinc in an metalloenzyme called
ALAD.
•
ALAD is responsible for making hemoglobin.
•
Without enough hemoglobin, brain does not get
enough oxygen.
•
Direct links between exposure to lead and reduced
cognitive abilities have been established
•
Studies show a relationship between lead
exposure and violent crime
Control of enzyme function:
allosteric regulation
• the enzyme usually flips back
and forth between active and
inactive forms by itself
• if activators are present, the
enzyme is stabilized in the
ACTIVE form
• if inhibitors are present, the
enzyme is stabilized in the
INACTIVE form
Control of enzyme
function:
feedback inhibition
• if there is enough product
present, the product can
stop (inhibit) the pathway by
acting as an inhibitor
• the product binds to the
enzyme at an allosteric site
and INACTIVATES the
enzyme
• this way, the pathway will
only make more product if
there is a need to make more
cooperativity
• sometimes, if there is an enzyme that can bind multiple substrates, the
binding of one substrate molecule can cause the whole enzyme to be
ACTIVATED and more receptive to the other substrates
• ex: hemoglobin can bind 4 oxygen molecules  if one oxygen binds
first, this makes the enzyme able to bind the other oxygens more easily
Kinetics of Enzyme Activity
• Substrate concentration affects activity
– usually expressed using a Michaelis-Menton
plot,
– enzymes which generate such a plot are said
to obey Michaelis-Menton kinetics
What is Vmax?
•
•
•
•
Vmax is maximum rate for an enzyme
catalyzed reaction can
It is a limit of the enzyme.
At the point when all of the active sites
are engaged with substrate, Vmax has
been reached.
Only increasing the amount of enzyme
will increase Vmax
Kinetics of Enzyme Activity
• E + S  ES  P + E
• Michaelis and Menton
studied enzyme kinetics
– Results: an easier way to
determine enzyme catalyzed
reaction rate:
– Km = Michaelis-Menton
constant
•
•
•
•
E = enzyme
S = substrate
ES = enzyme-subrate complex
P = Product
V
[S
]
m
a
x
r
a
te
v
• mixture of rate constants for
K

[S
]
forward and reverse rxns
m
– Vmax = theoretical max rate
limited by amount of enzyme.
Michaelis-Menton Plot
• 3 regions
– Low [S]: First order
wrt to [S]
– High [S]: Zero order,
b/c rxn rate is limited
by amount of
enzyme
– Middle [S]: mixed
order
What is Km?
• Mixture
of
rate
constants
k
–E
1
k2
ES
k3
k2
k3
k
P+E
• Km is independent of the substrate
concentration
• Experimentally determined
• Value varies with kind of substrate, temp
and pH
• Higher Km = lower enzyme activity
• At low [S]:
– [S] << Km
– Rate is proportional to
substrate conc. As
expected for first order
• At high [S]
– [S] >> Km
– Rate = Vmax
V
[S
]
m
a
x
r
a
te
v
K

[S
]
m
Determining Km and Vmax
• If …
V
[S
]
m
a
x
r
a
te
v
K

[S
]
m
• When v = ½ Vmax
• Km = [S]
– So you can read
this from the
Michaelis-Menton
Plot
• Which enzyme
has a lower Km?
– Hexokinase
• Which enzyme
has a higher
affinity for
glucose?
– Hexokinase
• So: lower Km =
faster rate
There is only a small amount of hexokinase in the blood. It is very
sensitive to glucose. When glucose concentrations are high,
glucokinase, of which there is much more, can pick up the slack.
• Why have 2
enzymes for
converting
sugar?
• At high
[glucose]:
– glucokinase
can speed up
rapidly storing
glucose in liver
Another way: Lineweaver-Burke Plot
• Take the inverse of both sides:
V
[S
]
m
a
x
r
a
te
v
K

[S
]
m

1 K
1
m 1

  
v
V
[]
S V
m
a
x
m
a
x
y=
m
• Graph of 1/rate vs. 1/[S] gives a line
– Slope = Km/Vmax
– Y-intercept = 1/Vmax
x+
b
Lineweaver-Burke Plot
• Graph of 1/rate vs. 1/[S] gives a line
– Slope = Km/Vmax
– Y-intercept = 1/Vmax
Competitive Inhibitor
• Competitive Inhibition:
– Takes more [S] to
overcome competitive
inhibitor, but eventually
it is overcome.
– Km changes b/c it
appears that the
enzyme has less
affinity for the
substrate, Vmax stays
the same
Non-competitive Inhibitor
• Reduced number of
active sites
– Vmax changes
• Km stays the same
– because for the enzyme
that works, there is the
same affinity for the
substrate as their was
when the non-competitive
inhibitor was present.
There is just less enzyme.
• Non-competitive inhibition
cannot be overcome by
adding more substrate
• http://wiz2.pharm.wayne.edu/biochem/enz.
html