Enzyme Mechanisms and Regulation

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Enzyme Mechanisms and Regulation

• Andy HowardIntroductory Biochemistry, Fall
  2008Tuesday 28 October 2008

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How do enzymes reduce activation energies?

• We can illustrate mechanistic principles by
  looking at specific examples we can also
  recognize enyzme regulation when we see it.

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Mechanism Topics

• Regulation
• Thermodynamics
• Enzyme availability
• Allostery, revisited

• Mechanisms
• Induced-fit
• Tight Binding of Ionic Intermediates
• Serine proteases
• Other proteases
• Lysozyme

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Examining enzyme mechanisms will help us
understand catalysis

• Examining general principles of catalytic
  activity and looking at specific cases will
  facilitate our appreciation of all enzymes.

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Binding modes proximity

• We describe enzymatic mechanisms in terms of the
  binding modes of the substrates (or, more
  properly, the transition-state species) to the
  enzyme.
• One of these involves the proximity effect, in
  which two (or more) substrates are directed down
  potential-energy gradients to positions where
  they are close to one another. Thus the enzyme is
  able to defeat the entropic difficulty of
  bringing substrates together.

William Jencks
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Binding modes efficient transition-state binding

• Transition state fits even better (geometrically
  and electrostatically) in the active site than
  the substrate would. This improved fit lowers the
  energy of the transition-state system relative to
  the substrate.
• Best competitive inhibitors of an enzyme are
  those that resemble the transition state rather
  than the substrate or product.

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Proline racemase

• Pyrrole-2-carboyxlate resembles planar transition
  state

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Yeast aldolase

• Phosphoglycolohydroxamate binds much like the
  transition state to the catalytic Zn2

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Adenosine deaminase with transition-state analog

• Transition-state analogKi10-8 substrate Km
• Wilson et al (1991) Science 252 1278

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ADA transition-state analog

• 1,6 hydrate of purine ribonucleoside binds with
  KI 310-13 M

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Induced fit

• Refinement on original Emil Fischer lock-and-key
  notion
• both the substrate (or transition-state) and the
  enzyme have flexibility
• Binding induces conformational changes

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Example hexokinase

• Glucose ATP ? Glucose-6-P ADP
• Risk unproductive reaction with water
• Enzyme exists in open closed forms
• Glucose induces conversion to closed form water
  cant do that
• Energy expended moving to closed form

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Hexokinase structure

• Diagram courtesy E. Marcotte, UT Austin

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Tight binding of ionic intermediates

• Quasi-stable ionic species strongly bound by
  ion-pair and H-bond interactions
• Similar to notion that transition states are the
  most tightly bound species, but these are more
  stable

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Serine protease mechanism

• Only detailed mechanism that well ask you to
  memorize
• One of the first to be elucidated
• Well studied structurally
• Illustrates many other mechanisms
• Instance of convergent and divergent evolution

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The reaction

• Hydrolytic cleavage of peptide bond
• Enzyme usually works on esters too
• Found in eukaryotic digestive enzymes and in
  bacterial systems
• Widely-varying substrate specificities
• Some proteases are highly specific for particular
  aas at position 1, 2, -1, . . .
• Others are more promiscuous

CH
NH
C
NH
C
NH
R1
CH
O
R-1
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Mechanism

• Active-site serine OH Without neighboring
  amino acids, its fairly non-reactive
• becomes powerful nucleophile because OH proton
  lies near unprotonated N of His
• This N can abstract the hydrogen at near-neutral
  pH
• Resulting charge on His is stabilized by its
  proximity to a nearby carboxylate group on an
  aspartate side-chain.

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Catalytic triad

• The catalytic triad of asp, his, and ser is found
  in an approximately linear arrangement in all the
  serine proteases, all the way from non-specific,
  secreted bacterial proteases to highly regulated
  and highly specific mammalian proteases.

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Diagram of first three steps
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Diagram of last four steps
Diagrams courtesy University of Virginia
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Chymotrypsin as example

• Catalytic Ser is Ser195
• Asp is 102, His is 57
• Note symmetry of mechanismsteps read similarly
  L? R and R ? L

Diagram courtesy of Anthony Serianni, University
of Notre Dame
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Oxyanion hole

• When his-57 accepts proton from Ser-195it
  creates an RO- ion on Ser sidechain
• In reality the Ser O immediately becomes
  covalently bonded to substrate carbonyl carbon,
  moving - charge to the carbonyl O.
• Oxyanion is on the substrate's oxygen
• Oxyanion stabilized by additional interaction in
  addition to the protonated his 57main-chain NH
  group from gly 193 H-bonds to oxygen atom (or
  ion) from the substrate,further stabilizing the
  ion.

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Oxyanion hole cartoon

• Cartoon courtesy Henry Jakubowski, College of
  St.Benedict / St.Johns University

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Modes of catalysis in serine proteases

• Proximity effect gathering of reactants in steps
  1 and 4
• Acid-base catalysis at histidine in steps 2 and 4
• Covalent catalysis on serine hydroxymethyl group
  in steps 2-5
• So both chemical (acid-base covalent) and
  binding modes (proximity transition-state) are
  used in this mechanism

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Specificity

• Active site catalytic triad is nearly invariant
  for eukaryotic serine proteases
• Remainder of cavity where reaction occurs varies
  significantly from protease to protease.
• In chymotrypsin ? hydrophobic pocket just
  upstream of the position where scissile bond sits
• This accommodates large hydrophobic side chain
  like that of phe, and doesnt comfortably
  accommodate hydrophilic or small side chain.
• Thus specificity is conferred by the shape and
  electrostatic character of the site.

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Chymotrypsin active site

• Comfortably accommodates aromatics at S1 site
• Differs from other mammalian serine proteases in
  specificity

Diagram courtesy School of Crystallography,
Birkbeck College
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Divergent evolution

• Ancestral eukaryotic serine proteases presumably
  have differentiated into forms with different
  side-chain specificities
• Chymotrypsin is substantially conserved within
  eukaryotes, but is distinctly different from
  elastase

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iClicker quiz!

• Why would the nonproductive hexokinase reaction
  H2O ATP -gt ADP Pibe considered
  nonproductive?
• (a) Because it needlessly soaks up water
• (b) Because the enzyme undergoes a wasteful
  conformational change
• (c) Because the energy in the high-energy
  phosphate bond is unavailable for other purposes
• (d) Because ADP is poisonous
• (e) None of the above

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iClicker quiz, question 2Why are proteases
often synthesized as zymogens?

• (a) Because the transcriptional machinery cannot
  function otherwise
• (b) To prevent the enzyme from cleaving peptide
  bonds outside of its intended realm
• (c) To exert control over the proteolytic
  reaction
• (d) None of the above

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Question 3 what would bind tightest in the TIM
active site?

• (a) DHAP (substrate)
• (b) D-glyceraldehyde (product)
• (c) 2-phosphoglycolate(Transition-state analog)
• (d) They would all bind equally well

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Convergent evolution

• Reappearance of ser-his-asp triad in unrelated
  settings
• Subtilisin externals very different from
  mammalian serine proteases triad same

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Subtilisin mutagenesis

• Substitutions for any of the amino acids in the
  catalytic triad has disastrous effects on the
  catalytic activity, as measured by kcat.
• Km affected only slightly, since the structure of
  the binding pocket is not altered very much by
  conservative mutations.
• An interesting (and somewhat non-intuitive)
  result is that even these "broken" enzymes still
  catalyze the hydrolysis of some test substrates
  at much higher rates than buffer alone would
  provide. I would encourage you to think about why
  that might be true.

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Cysteinyl proteases

• Ancestrally related to ser proteases?
• Cathepsins, caspases, papain
• Contrasts
• Cys SH is more basicthan ser OH
• Residue is less hydrophilic
• S- is a weaker nucleophile than O-

Diagram courtesy ofMariusz Jaskolski,U. Poznan
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Papain active site
Diagram courtesy Martin Harrison,Manchester
University
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Hen egg-white lysozyme

• Antibacterial protectant ofgrowing chick embryo
• Hydrolyzes bacterial cell-wall peptidoglycans
• hydrogen atom of structural biology
• Commercially available in pure form
• Easy to crystallize and do structure work
• Available in multiple crystal forms
• Mechanism is surprisingly complex (14.7)

HEWLPDB 2vb10.65Å15 kDa
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Mechanism of lysozyme

• Strain-induced destabilization of substrate makes
  the substrate look more like the transition state
• Long arguments about the nature of the
  intermediates
• Accepted answer covalent intermediate between
  D52 and glycosyl C1 (14.39B)

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The controversy
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Regulation of enzymes

• The very catalytic proficiency for which enzymes
  have evolved means that their activity must not
  be allowed to run amok
• Activity is regulated in many ways
• Thermodynamics
• Enzyme availability
• Allostery
• Post-translational modification
• Protein-protein interactions

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Thermodynamics as a regulatory force

• Remember that ?Go is not the determiner of
  spontaneity ?G is.
• Therefore local product and substrate
  concentrations determine whether the enzyme is
  catalyzing reversible reactions to the left or to
  the right
• Rule of thumb ?Go lt -20 kJ mol-1 is irreversible

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Enzyme availability

• The enzyme has to be where the reactants are in
  order for it to act
• Even a highly proficient enzyme has to have a
  nonzero concentration
• How can the cell control Etot?
• Transcription (and translation)
• Protein processing (degradation)
• Compartmentalization

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Transcriptional control

• mRNAs have short lifetimes
• Therefore once a protein is degraded, it will be
  replaced and available only if new
  transcriptional activity for that protein occurs
• ? Many types of transcriptional effectors
• Proteins can bind to their own gene
• Small molecules can bind to gene
• Promoters can be turned on or off

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Protein degradation

• All proteins havefinite half-lives
• Enzymes lifetimes often shorter than structural
  or transport proteins
• Degraded by slings arrows of outrageous
  fortune or
• Activity of the proteasome, a molecular machine
  that tags proteins for degradation and then
  accomplishes it

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Compartmentalization

• If the enzyme is in one compartment and the
  substrate in another, it wont catalyze anything
• Several mitochondrial catabolic enzyme act on
  substrates produced in the cytoplasm these
  require elaborate transport mechanisms to move
  them in
• Therefore, control of the transporters confers
  control over the enzymatic system

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Allostery

• Remember we defined this as an effect on protein
  activity in which binding of a ligand to a
  protein induces a conformational change that
  modifies the proteins activity
• Ligand may be the same molecule as the substrate
  or it may be a different one
• Ligand may bind to the same subunit or a
  different one
• These effects happen to non-enzymatic proteins as
  well as enzymes

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Substrates as allosteric effectors (homotropic)

• Standard example binding of O2 to one subunit of
  tetrameric hemoglobin induces conformational
  change that facilitates binding of 2nd ( 3rd
  4th) O2s
• So the first oxygen is an allosteric effector of
  the activity in the other subunits
• Effect can be inhibitory or accelerative

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Other allosteric effectors (heterotropic)

• Covalent modification of an enzyme by phosphate
  or other PTM molecules can turn it on or off
• Usually catabolic enzymes are stimulated by
  phosphorylation and anabolic enzymes are turned
  off, but not always
• Phosphatases catalyze dephosphorylation these
  have the opposite effects

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Cyclic AMP-dependent protein kinases

• Enzymes phosphorylate proteins with S or T within
  sequence R(R/K)X(S/T)
• Intrasteric controlregulatory subunit or domain
  has a sequence that looks like the target
  sequence this binds and inactivates the kinases
  catalytic subunit
• When regulatory subunits binds cAMP, it releases
  from the catalytic subunit so it can do its thing

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Kinetics of allosteric enzymes

• Generally these dont obey Michaelis-Menten
  kinetics
• Homotropic positive effectors produce sigmoidal
  (S-shaped) kinetics curves rather than hyperbolae
• This reflects the fact that the binding of the
  first substrate accelerates binding of second and
  later ones

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T ? R State transitions

• Many allosteric effectors influence the
  equilibrium between two conformations
• One is typically more rigid and inactive, the
  other is more flexible and active
• The rigid one is typically called the tight or
  T state the flexible one is called the
  relaxed or R state
• Allosteric effectors shift the equilibrium toward
  R or toward T