Table of Contents
Fundamentals of Biochemistry
Biochem 380 - Fall 2006
Lecture 015
Outline
- Quiz
- Announcements
- Questions from previous lecture
- Section 5.8: Irreversible Inhibition
- Section 5.9: Allosteric Enzymes
- Section 5.10: Enzyme Regulation
- Section 5.11: Multi-Enzyme Complexes
Announcements
- Lecture 14 notes are online
- Reminder: Midterm 1 is this Thursday, SC/CHCP 131, 6:00pm-8:00pm, on Chapters 1-5
- Proposed review session on Wednesday evening with Chris
Questions
- Any questions on the material from the previous lecture?
Section 5.8: Irreversible Inhibition
- Like reversible inhibition, irreversible inhibition reduces the activity of an enzyme.
- However, as the name implies, it is more permanent because it usually involves the formation of a covalent bond
between the inhibitor and the enzyme
- Often, the reaction is an alkylation or acylation of a side chain of an amino acid residue
in the enzyme
- A common feature of many irreversible inhibitors is that they resemble the natural substrate of
the enzyme, so that they bind as substrate analogs
- In addition, they often form bonds with especially reactive groups in the active site of an enzyme
(eg: the catalytic serine residue of a serine protease)
- Consequently, these kinds of inhibitors are found in toxins and can be used
for analytical purposes
Covalent Modification of Serine
- Diisopropyl fluorophosphate (DFP) is a nerve gas that forms a covalent bond with a
reactive serine residue found in many protease and esterase enzymes
- In the active site of chymotrypsin, shown above, the hydroxyl group of Ser195 attacks the
phosphorus atom in DFP, forming a phosphoryl linkage that inactivates the enzyme
- In addition to identifying active site residues for analytical purposes, organic phosphorus
compounds such as DFP were developed as toxic agents for military purposes, and also for use
as insecticides
Covalent Modification of Glutamate
- Other irreversible inhibitors have particular structures that allow them to bind specifically
to the active site of an enzyme
- Bromohydroxyacetone phosphate (BHAP) is an analog of dihydroxyacetone phosphate (DHAP), which binds
to the active site of triose phosphate isomerase (an enzyme we'll see later in glycolysis)
- In the reaction, the bromine group on the inhibitor leaves, resulting in the formation of
a covalent bond to the active Glu residue on the enzyme
Covalent Modification of Lysine
- Lysine residues are another target for irreversible inhibitors. The free ε-amino group
at the end of the side chain can react with an aldehyde to form a Schiff Base, which is characterized by
a double bond between the carbonyl carbon and the amine nitrogen
- The resulting base can then be reduced by sodium borohydride (NaBH4) to produce
a stable substituted enzyme
- If an assay shows a subsequent loss of activity, then this implicates the lysine residue as being located
in the active site of the enzyme
Acetylcholinesterase Inhibitors and Alzheimers
- Alzheimers Disease (AD) is the most common form of dementia and a leading cause of death in developed nations.
It is associated with neuronal death, accumulation of deposits (plaques) and lowering of acetylcholine (ACh)
levels in the cerebral cortex
- The cause of AD is still unknown, and effective therapies are not yet available. However, a number of drugs have
been approved recently to treat the symptoms of the disease, which include memory loss and other cognitive deficits
- These drugs, which include donepezil (Aricept) and tacrine (Cognex), act as inhibitors of
acetylcholinestrase (AChE)
- AChE is an enzyme that breaks down acetylcholine (ACh), one of the principal neurotransmitters in the central
nervous system
AChE Inhibitors
- These are examples of quasi-irreversible inhibitors, which can bind to the enzyme for long periods
of time (up to 10 hours, for donepezil)
- The inhibition of AChE prevents it from cleaving the ester linkage in ACh, allowing the concentration
of the neurotransmitter to be maintained at higher levels and increasing neuronal activity
- In addition to binding at the active site, Donepezil also binds to the Peripheral Anionic Site (PAS)
on AChE, a secondary site associated with β-amyloid aggregation
Section 5.9: Allosteric Enzymes
- The allosteric properties of enzymes allow them to function somewhat akin to transistors in the chemical
control systems of the cell
- This capability for control derives from the ability of a small molecule to bind to the enzyme,
as an input that involves only a very small amount of energy
- The consequence of binding, or output, is to effect the catalytic activity of the enzyme,
which typically involves much greater energies
- This 'switching' effect of allosteric interaction results from the conformational changes that
occur on binding. Such changes can occur both on single-subunit enzymes, and more commonly, on
multisubunit complexes where the relative positions and orientations between subunits are altered on binding
- The allosteric activity of an enzyme can be seen from its kinetics, where the velocity curve is typically
sigmoidal in nature (non-Michaelis-Menten kinetics)
Properties of Allosteric Enzymes
- The graph above shows how the velocity of enzyme catalysis varies with allosteric regulation
- The blue curve represents an enzyme with an activator bound, so that it is stabilized in the active 'R' state.
Consequently, the curve is hyperbolic, similar to Michaelis-Menten kinetics
- Without activator, and with increasing levels of an inhibitor, the curve changes to a sigmoidal shape,
where smaller concentrations of substrate are associated with the enzyme being in the inactive 'T' state
Phosphofructokinase
- Phosphofructokinase is one of the enzymes involved in glycolysis and represents one of the principle
points of control for the pathway
- It catalyzes the transfer of a phosphoryl group from ATP to fructose-6-phosphate to produce
fructose 1,6-bisphosphate
- The reaction represents one of the committed steps in glycolysis and so it is one of the
main regulatory enzymes of the pathway, with multiple allosteric effectors
Structure of Phosphofructokinase
- The structure of phosphofructokinase (PFK) is shown above. To the left is the monomer, which includes
the active site with products fructose 1,6-bisphosphate (F1,6-BP) and ADP
- At the bottom left is the allosteric site which can bind two different regulators,
ADP and phosphoenolpyruvate (PEP)
- When ADP binds in the regulator site, it an activator, stabilizing PFK in the R state
- When PEP binds, it excludes ADP and acts as an inhibitor that stabilizes PFK in the T state
Models of Allosteric Regulation
- There are two main models that attempt to describe the cooperative effects of allosteric binding
for multimeric enzymes
- The two models are the concerted (MWC) model and the sequential (Koshland) model
- Both models attempt to provide a quantitative description of changes from the T to the R state
for all subunits in a complex, with varying degrees of success, depending upon the particular enzyme
- For many enzymes, the behavior is complex and can only be adequately described by combining
aspects from both models
The Concerted Model
- In the concerted model, the conformation of all substrates are assumed to change simultaneously
from the T to R state, with the R state becoming more stable with each additional bound substrate
The Sequential Model
- In the sequential model, which is more general, the conformation of each subunit can change individually
from T to R, and the subunit conformation can influence the adjacent subunits to varying degrees
The Composite Model for Hemoglobin
- Hemoglobin, because it is actually a dimer of two dimers rather than a tetramer, undergoes a transition
involving intermediate states where each dimer becomes fully bound with substrate
Section 5.10: Enzyme Regulation
- As there are a large number and variety of enzymes, so to is there a diversity of mechanisms for regulating
their activity. Regulation of enzymes include the following categories:
- Enzyme synthesis and degradation
- Isozymes
- Allosteric regulation
- Reversible covalent modification
- Proteolytic cleavage
Phosphorylation
- The most common type of reversible covalent modification is phosphorylation,
which involves the addition of a phosphoryl group on to the residues of enzymes
- For the most part, phosphoryl groups are added to to Ser, Thr and Tyr residues, transferred from ATP
- The enzymes which catalyze this phosphorylation are kinases. The importance of kinases
in regulation is demonstrated by the existence of over 500 different protein kinases in humans
(1.7% of genome). Collectively, they represent the 'kinome'
- Kinases regulate many kinds of activity in cells, including metabolism, signal transduction, cell division
and apoptosis
- The modification is reversible, through the complementary action of phosphatases, which are
enzymes that catalyze the removal of the phosphoryl groups
Section 5.11: Multi-Enzyme Complexes
- Many enzymes exist as parts of multi-enzyme complexes, where a sequence of catalyzed reactions are
associated
- Such complexes can increase the efficiency of the overall pathway, because products of one reaction
can rapidly bind as substrates of the next reaction in a sequence, instead of diffusing away
- In some cases, a complex may actually include a spatial tunnel to channel the product from
one enzyme active site to another
- An example of this is seen with tryptophan synthase, which catalyzes the final steps of
tryptophan synthesis (Section 17.3F). The structure of the enzyme contains a tunnel to guide the
indole ring to the active site of the final reaction, where it is exchanged with the side chain of serine
References
- Chance and Necessity, Jacques Monod,
(A biography of one of the first scientists to propose the allosteric nature of enzymes)
- From Enzymatic Adaption to Allosteric Transitions, Jacques Monod,
http://nobelprize.org/nobel_prizes/medicine/laureates/1965/monod-lecture.html
(Monod's Nobel lecture, 1965)
- The Protein Kinase Complement of the Human Genome,
G. Manning, D. B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam1,
6 Dec 2002 Vol 298 Science
- An Overview of the Current and Novel Drugs for Alzheimer’s Disease with
Particular Reference to Anti-Cholinesterase Compounds,
Marcela Colombres, Juan Paulo Sagal and Nibaldo C. Inestrosa,
Current Pharmaceutical Design, 2004, 10, 3121-3130
Questions
- Questions about the material covered today?
Next Lecture: Sections 6.1 - 6.3