Download Enzymes lecture 2

Enzymes
Introduction
Headlines of the day….
TRPM2: a multifunctional ion channel for calcium signalling
Published online before print December 6, 2010, doi: 10.1113/jphysiol.2010.201855 J Physiol April 1, 2011 vol. 589 no. 7
1515-1525
Figure 2. Signalling mechanisms for TRPM2 activation NAD+ and reactive oxygen species (ROS), including H2O2, accumulate
during inflammation and tissue damage. External NAD+ may be converted to ADPR, cADPR and NAADP by the ectoenzymes
CD38 and CD157. Extracellular ADPR may then bind to plasma membrane receptors (e.g. P2Y receptors) and increases
[Ca2+]i through Ca2+ release from stores via G-proteins and phospholipase C (PLC) activation with subsequent IP3 production.
H2O2 may also cross the plasma membrane and mobilize ADPR from mitochondria (both H2O2 and cADPR can synergize with
ADPR to activate TRPM2). ADPR is also generated from poly-ADPR during ROS-induced DNA damage through activation of
the PARP-1/PARG pathway. Free cytosolic ADPR will act on the NUDT9-H of lysosomal and plasma membrane TRPM2
channels, enabling Ca2+ influx across the plasma membrane and/or release of lysosomal Ca2+, raising the Ca2+ concentration
in the cytosol. Ca2+ overload can trigger programmed cell death (apoptosis) and possibly necrosis. Finally, extracellular
signals that remain to be identified could potentially induce the production of intracellular free ADPR, which may then gate
TRPM2 channels in the lysosome and/or plasma membrane and regulate receptor-mediated signalling.
Headlines of the day
• The membrane-bound enzyme CD38 exists in two opposing orientations.
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Zhao YJ, Lam CM, Lee HC.
Source:Department of Physiology, Li Ka Shing School of Medicine, The University of Hong Kong,
Hong Kong, China.
Abstract
The transmembrane enzyme CD38, a multifunctional protein ubiquitously present in cells, is the
main enzyme that synthesizes and hydrolyzes cyclic adenosine 5'-diphosphate-ribose (cADPR), an
intracellular Ca(2+)-mobilizing messenger. CD38 is thought to be a type II transmembrane protein
with its carboxyl-terminal catalytic domain located on the outside of the cell; thus, the mechanism
by which CD38 metabolizes intracellular cADPR has been controversial. We developed specific
antibodies against the amino-terminal segment of CD38 and showed that two opposing
orientations of CD38, type II and type III (which has its catalytic domain inside the cell), were both
present on the surface of HL-60 cells during retinoic acid-induced differentiation. When activated
by interferon-γ, human primary monocytes and the monocytic U937 cell line exhibited a similar codistribution pattern. Site-directed mutagenesis experiments showed that the membrane
orientation of CD38 could be converted from a mixture of type II and type III orientations to all type
III by mutating the cationic amino acid residues in the amino-terminal segment of CD38. Expression
of type III CD38 construct in transfected cells led to increased intracellular concentrations of cADPR,
indicating the importance of the type III orientation of CD38 to its Ca(2+) signaling function. The
identification of these two forms of CD38 suggests that flipping the catalytic domain from the
outside to the inside of the cell may be a mechanism regulating its signaling activity.
GENERAL CHARACTERISTICS
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Most enzymes are protein, of the globular form.
Being proteins, they are affected by extremes of heat, pH and radiation.
All enzymes speed up the rate of a reaction.
All enzyme catalyzed reactions are reversible.
Enzymes speed up the rate of reaction by lowering the energy of
activation, i.e. Ea.
Enzymes are not involved in the reaction.
They are not destroyed at the end of a reaction.
They are either intracellular or extracellular in function.
Enzymes work only in solution.
All enzyme catalyzed reactions are affected by substrate concentration
and enzyme concentration.
All enzymes are specific for their substrate.
How enzymes work?
• Enzymes attach only to their
specific substrate.
• They remain in an enzymesubstrate complex.
• They breakdown/build the
substrate into respective
products.
• Enzyme dissociates itself
from the product.
Activation energy
The energy that an atomic system must acquire before a process (such as an emission or
reaction) can occur; "catalysts are said to reduce the energy of activation during the
transition phase of a reaction."
Energonic and exergonic energy
Structure and mechanism
Structures and mechanisms Enzymes are generally globular proteins and range from 62 amino
acid residues in size, to over 2,500 residues in the animal fatty acid synthase. The activities of
enzymes are determined by their three-dimensional structure Most enzymes are much larger
than the substrates they act on, and only a small portion of the enzyme (around 3–4 amino
acids) is directly involved in catalysis.
Giorgio Lampis, Alessandra Desogus, Sabrina Petruzzelli, Samuela Laconi, Angela Ingianni, Maria Antonietta Madeddu,
Raffaello Pompe, Anaerobe, Volume 13, Issues 5–6, October–December 2007, Pages 238–243
Dalton…unit of mass
The dalton, symbol Da, is also sometimes used as a unit of molar mass,
especially in biochemistry, with the definition 1 Da = 1 g/mol, despite the
fact that it is strictly a unit of mass (1 Da = 1.660 538 782(83)×10−27 kg.)
Maltase enzyme
Maltose is made of two glucose molecules bonded together (1). The maltase enzyme is a
protein that is perfectly shaped to accept a maltose molecule and break the bond (2). The two
glucose molecules are released (3). A single maltase enzyme can break in excess of 1,000
maltose bonds per second, and will only accept maltose molecules.
Characteristics of Maltase from Baker's Yeast:
Molecular weight: 68,500 daltons.
pH Optimum: 7.0 - 7.5 using maltose as the substrate.
Inhibitors: Thiol blocking compounds, heavy metal ions, histidine, and certain amines. Tris
should not be used as a buffer due to its inhibitory effect.
Storage: Store at 2-8° C.
Active site
The region that contains the catalytic residues, binds the substrate,
and then carries out the reaction is known as the active site. Enzymes
can also contain sites that bind cofactors, which are needed for
catalysis. Some enzymes also have binding sites for small molecules,
which are often direct or indirect products or substrates of the
reaction catalyzed. This binding can serve to increase or decrease the
enzyme's activity, providing a means for feedback regulation.
Jesse saying no to Bessie…politely
The frequency of decreased lactase activity ranges from 5% in northern Europe through 71% for
Sicily to more than 90% in some African and Asian countries.
Denaturation and reversibility
Most enzymes can be denatured—that is, unfolded and
inactivated—by heating, which destroys the three-dimensional
structure of the protein. Depending on the enzyme, denaturation
may be reversible or irreversible.
Models of enzyme action
"Lock and key" model :
"Lock and key" model Enzymes are very specific, because
both the enzyme and the substrate possess specific
complementary geometric shapes that fit exactly into
one another. This is often referred to as "the lock and
key" model. However, while this model explains enzyme
specificity, it fails to explain the stabilization of the
transition state that enzymes achieve.
Induced fit model :
Induced fit model Diagrams to show the induced fit
hypothesis of enzyme action.
since enzymes are rather flexible structures, the active
site is continually reshaped by interactions with the
substrate as the substrate interacts with the enzyme. As
a result, the substrate does not simply bind to a rigid
active site. In some cases, such as glycosidases , the
substrate molecule also changes shape slightly as it
enters the active site.
Transition State Model
Transition State Theory:
In the transition state theory, the mechanism of interaction of reactants is not
considered; the important criterion is that colliding molecules must have sufficient
energy to overcome a potential energy barrier (the activation energy) to react.
It takes a lot of energy to achieve the transition state, so the state is a high-energy
substance. The potential energy of the system increases at this point because:
•The approaching reactant molecules must overcome the mutual repulsive forces
between the outer shell electrons of their constituent atoms.
•Atoms must be separated from each other as bonds are broken.
Lock and Key Model
P
+
S
S
+
P
E
+ S
ES complex
E + P
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Enzyme Action:
Lock and Key Model
• An enzyme binds a substrate in a region called the active
site
• Only certain substrates can fit the active site
• Amino acid R groups in the active site help substrate bind
• Enzyme-substrate complex forms
• Substrate reacts to form product
• Product is released
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Enzyme Action:
Induced Fit Model
P
S
S
P
E
+ S
ES complex
E + P
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Enzyme Action:
Induced Fit Model
• Enzyme structure flexible, not rigid
• Enzyme and active site adjust shape to bind
substrate
• Increases range of substrate specificity
• Shape changes also improve catalysis during
reaction
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Transition State Model
For a reaction involving two molecules,
a transition state is formed when the
�old bonds between two molecules are
weakened and new bonds begin to form
or the old bonds break first to form the
transition state and then the new bonds
form after. The theory suggests that as
reactant molecules approach each other
closely they are momentarily in a less
stable state than either the reactants or
the products. In the example below, the
first scenario occurs to form the
transition state: