Most biochemical reactions catalyzed by enzymes involve the reaction of two or more substrates, ofte

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Multisubstrate Reactions

• Most biochemical reactions catalyzed by enzymes
  involve the reaction of two or more substrates,
  often resulting in multiple products
•  
• example proteolysis
• two substrates
• polypeptide
• water
• two products
• the two fragments of the cleaved polypeptide
  chain
• example phosphorylation of glucose (catalyzed by
  hexokinase)
• two substrates
• Glucose
• ATP
• two products
• glucose-6-phosphate
• ADP
•  

bi-bi
bi-bi
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Sequential Displacement
All substrates bind before the reaction occurs
and substrate is released
Sequential Ordered one substrate must bind
before a second substrate can bind
significantly
lactate dehydrogenase
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Sequential Displacement cont
Sequential Random either substrate can bind first
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Non-sequential displacement Double-Displacement
(Ping-Pong)
Non-sequential one substrate is bound, one
product is released, a second substrate comes in
and a second product is released results
in a substituted enzyme intermediate (modified
enzyme)
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Enzyme Inhibition
Enzyme inhibitors molecules that interfere with
catalysis, slowing or halting enzymatic
reactions Broad classification reversible and
irreversible

• 3 types of reversible inhibition
• competitive
• non competitive (mixed)
• uncompetitive

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• Bind to the enzymes active site
• Molecule closely resembles the substrate for an
  enzyme and is accepted into the enzymes binding
  site, but catalysis does not occur
• An EI complex is formed (rather than an ES
  complex)

Competitive inhibitors
Competitive inhibitor diminishes the rate of
catalysis by reducing the proportion of enzyme
molecules bound to substrate
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Kinetics of competitive inhibition
In this experiment three concentrations of enzyme
inhibitor are added to increasing amounts of
substrate The enzyme concentration is
constant What is this experiment suggesting
regarding the effect of substrate concentration
on this particular inhibitor?
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Competitive Inhibition cont
Competitive Inhibitor competes with the substrate
for the active site Increasing amounts of
substrate can overcome this inhibition Can
sufficient substrate be added to achieve Vmax?
What is the effect of a competitive inhibitor on
Km? (enzyme cannot bind substrate as well when
inhibitor is present)
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Non-competitive inhibition
inhibitors bind at a site distinct from the
substrate active site on the enzyme, but will
bind to either E or ES
non-competitive inhibitor lowers the
concentration of functional enzyme
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Kinetics of non-competitive inhibition
In this experiment three concentrations of
inhibitor are added to increasing amounts of
substrate The enzyme concentration is
constant Will increasing concentrations of
substrate overcome non-competitive inhibition to
the point where Vmax will be achieved?
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Non-competitive inhibition
Non-competitive inhibitor binds to a site
distinct from the enzymes active site Increasing
amounts of substrate will not overcome this
inhibition so that Vmax will be achieved
What is the effect of a non-competitive inhibitor
on Km?
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Uncompetitive inhibitors
binds at a sight distinct from the substrate but
only in the ES complex (non competitive
inhibitor binds to either free enzyme or the ES
complex)
uncompetitive inhibition requires that the
inhibitor affect the catalytic function of an
enzyme but not its substrate binding
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Comparison of competitive and non-competitive
inhibition
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Enzyme Inhibition-Irreversible inhibitors
Enzyme inhibitors molecules that interfere with
catalysis, slowing or halting enzymatic
reactions Broad classification reversible and
irreversible

• 3 types of reversible inhibition
• competitive
• non competitive (mixed)
• uncompetitive

Irreversible inhibitors covalently combine with
or destroy a functional group on an enzyme that
is essential for the enzymes activity, or that
form a particularly stable noncovalent
association that does the same thing
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Most irreversible inhibitors are toxic substances
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Irreversible inhibitors as a tool for studying
reaction mechanism
Amino acids in the active site that are involved
in catalysis can sometimes be identified by
determining which amino acid is covalently linked
to an inhibitor (complements X-ray
crystallography)

• Irreversible inhibitors
• group specific reagents
• substrate analogs
• suicide inhibitors

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Irreversible inhibitors group specific reagents
Group specific reagents react with specific R
groups
There are 27 serines in chymotrypsin DIFP
(disopropylfluorphosphate) irreversibly inhibits
chymotrypsin and turns out to modify serine 195
indicating this serine is reactive with substrate
DIFP also binds to a reactive serine in
acetylcholinesterase and inactivates the enzyme
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Irreversible inhibitors substrate analogs
substrate analogs (affinity labels) are more
specific for the enzyme active site than group
specific reagents
TPCK binds irreversibly with His57 in the enzyme
active site
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Irreversible inhibitors suicide inhibitors
Suicide inhibitors (mechanism-based
inactivators) more specific than group-specific
or substrate analogs binds to the enzyme as a
substrate, is catalyzed, but instead of being
transformed into the normal product, is converted
to a very reactive compound that then combines
irreversibly with the enzyme
Enzyme is monamine oxidase FAD is a prosthetic
group which oxidizes the inhibitor The oxidized
inhibitor then covalently modifies FAD by
alkylating N-5
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Monoamine Oxidase Inhibitors
When monoamine oxidases deaminate
neurotransmitters such as dopamine and serotonin
what is the effect on the levels of these
neurotransmitters?
Depression is associated with low levels of
serotonin Parkinson disease is associated with
low levels of dopamine
What would the expected result of MAO inhibitors
be on levels of serotonin and dopamine?
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Transition state analogs as enzyme inhibitors
If enzymes are structurally complementary to the
transition state then some functional groups in
both the substrate and the enzyme must react
preferentially in the transition state (instead
of the ES complex)
In the following experiment substrate A has been
modified Do substrates B and C still bind to the
enzyme? (chymotrypsin which normally catalyzes
the hydrolysis of peptide bonds next to aromatic
amino acids) The impact on kcat and kcat/km
suggests that the transition state is stabilized
Pharmaceuticals that are transition state analogs
can bind an enzyme more tightly than the normal
substrate for the enzyme (protease inhibitor for
HIV) In principle, they should fit into the
substrate binding site better (there should be a
greater number of weak interactions in the
transition state then in the enzyme substrate
complex)
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Catalytic Antibodies
What happens when you use a transition state
analog to generate an antibody? What will the
antibody bind to?
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Biochemical example of enzyme inhibition
Penicillin (and related analogs)
Peptidoglycans give bacterial cell walls their
rigidity and are unique to bacterial cell walls
no homologous structure exists in
mammals Penicillins are structural analogs of
substrates of the transpeptidases and block
formation of the peptide cross-links in
peptidoglycans (via suicide inhibition an
irreversible inhibition)
Penicillin and its analogues all contain a
b-lactam ring with a thiazolidine ring attached
(but differ in the substituent at C-6 (which
accounts for the different pharmacological
properties) example pen V is acid stable and
can be administered orally, but methicillin is
acid labile and must be given intravenously or
intramuscularly
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Penicillin and antibiotic resistance

• b-lactamase (penicillinase) is an enzyme in
  bacteria that opens the lactam ring, rendering
  the compound harmless to the bacterium
• penicillin was first introduced as an antibiotic
  during WWII and at that time none of the major
  disease-causing bacteria possessed the
  b-lactamase gene

• How did bacteria acquire the b-lactamase gene?
•    conjugation (DNA is passed from one bacterial
  cell to another
•    transduction ( a bacterial gene is carried
  from cell to cell by a virus)
• transformation (a bacterial cell picks up
  naked DNA from its surrounding medium)

Pharmacological approach to the spread of
b-lactamase is the development of penicillin
derivatives (for example cefuroxime) that are
more resistant to hydrolysis by b-lactamase
(however, natural selection leads rapidly to the
evolution of new forms of b-lactamase) another
approach is to treat patients with a penicillin
derivative and a b-lactamase inhibitor a single
base change in a gene encoding a bacterial
b-lactamase may render useless 100 million
dollars worth of pharmaceutical research effort.
Other mechanisms of antibiotic resistance not
all penicillin-resistant bacteria have acquired
the b-lactamase gene         some are resistant
because they possess modifications in their cell
walls that block entry of the antibiotic        
some are able to selectively export the
antibiotic once it has entered the cell        
some possess modified transpeptidases that fail
to bind the antibiotic example bacterial
meningitis (Neisseria meningitides)
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Antibiotic resistance - continued
Vancomycin

• mechanism inhibits transpeptidation by binding
  to the peptide substrate rather than the enzyme
• normal substrate terminates in D-alanineD-alanine
  dipeptide
• resistant bacteria synthesize an alternative
  terminus that doesnt bind vancomycin
• this involves the acquisition of several new
  enzyme activities
• as a result vancomycin is the antibiotic to which
  bacteria have been least able to develop
  resistance to and is usually given as a last
  resort when other antibiotics have failed
• however, vancomycin resistant strains of several
  pathogenic bacteria have developed (Enterococcus
  faecium and Staphylococcus aureus)
• these resistant strains often reside in hospital
  wards, making hospital acquired infections
  increasingly difficult to treat)
• the selection for vancomycin-resistant strains is
  thought to have been driven by the wide-spread
  use of a related antibiotic (avoparcin) in animal
  feed
• currently researchers are attempting to modify
  the chemical structure of vancomycin to produce
  derivatives that may be effective to resistant
  bacteria

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Drug resistance and AIDS

• bacterial DNA replicating enzymes operate at a
  very high level of accuracy
• replicating enzyme of HIV (reverse transcriptase)
  is not as accurate and the errors in
  transcription lead to a high rate of mutation
• the high error rate combined with the high rate
  of virus production, makes it very likely that
  drug-resistant variants will emerge within an
  individual as the infection progresses
•  
• Approaches to drug resistance of HIV
• patients take several different drugs with
  different targets reducing the likelihood that a
  variant will emerge that is resistant to all of
  the drugs
• designing drugs that interact with the most
  highly conserved portions of targeted enzymes

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