REGULATION OF ENZYME ACTIVITY

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REGULATION OF ENZYME ACTIVITY

• Medical Biochemistry, Lecture 25

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Lecture 25, Outline

• General properties of enzyme regulation
• Regulation of enzyme concentrations
• Allosteric enzymes and feedback inhibition
• Other effectors of catalytic activity

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Metabolic Homeostasis
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General Properties Regulatory Enzymes

• The biochemical pathways that you will soon be
  studying are composed of groups of coordinated
  enzymes that perform a specific metabolic
  process. In general, these enzyme groups are
  composed of many enzymes, only a few of which are
  regulated by the mechanisms described in this
  lecture. Regulatory enzymes are usually the
  enzymes that are the rate-limiting, or committed
  step, in a pathway, meaning that after this step
  a particular reaction pathway will go to
  completion.

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General Properties Regulatory Enzymes (cont)

• Frequently, regulatory enzymes are at or near the
  initial steps in a pathway, or part of a branch
  point or cross-over point between pathways (where
  a metabolite can be potentially converted into
  several products in different pathways). In
  general, a cell needs to conserve energy -
  therefore costly (in metabolic terms)
  biosynthetic reaction pathways will not be
  operational unless a particular metabolite is
  required at a given time.

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General Properties Regulatory Enzymes (cont)

• Recall that when acting as catalysts, enzyme
  mediated-reactions should be reversible.
  However, regulatory enzymes frequently catalyze
  thermodynamically irreversible reactions, that
  is, a large negative free energy change (-DG)
  greatly favors formation of a given metabolic
  product rather than the reverse reaction. Thus,
  regulation of enzyme activity, usually at the
  committed step of the pathway, is critical for
  supplying and maintaining cellular metabolitic
  and energy homeostasis.

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Two General Mechanisms that Affect Enzyme
Activity

• 1) control of the overall quantities of enzyme or
  concentration of substrates present
• 2) alteration of the catalytic efficiency of the
  enzyme

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Regulation of Enzyme Concentrations

• The overall synthesis and degradation of a
  particular enzyme, also termed its turnover
  number, is one way of regulating the quantity of
  an enzyme. The amount of an enzyme in a cell can
  be increased by increasing its rate of synthesis,
  decreasing the rate of its degradation, or both.

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Regulation of Enzyme Concentrations Induction

• Induction (an increase caused by an effector
  molecule) of enzyme synthesis is a common
  mechanism - this can manifest itself at the level
  of gene expression, RNA translation, and
  post-translational modifications. The actions of
  many hormones and/or growth factors on cells will
  ultimately lead to an increase in the expression
  and translation of "new" enzymes not present
  prior to the signal. These generalizations will
  be covered in more detail in Dr. Bannon's
  lectures.

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Regulation of Enzyme Concentrations Degradation

• The degradation of proteins is constantly
  occuring in the cell, yet the molecular
  mechanisms that determine when and which enzymes
  will be degraded are poorly understood. The
  turnover number of an enzyme can be used for
  general comparison with other enzymes or other
  enzyme systems, yet these numbers can vary from
  minutes to hours to days for different enzymes.

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Regulation of Enzyme Concentrations Degradation
(cont)

• Protein degradation by proteases is
  compartmentalized in the cell in the lysosome
  (which is generally non-specific), or in
  macromolecular complexes termed proteasomes.
  Degradation by proteasomes is regulated by a
  complex pathway involving transfer of a 76 aa
  polypeptide, ubiquitin, to targeted proteins.
  Ubiquination of protein targets it for
  degradation by the proteasome. This pathway is
  highly conserved in eukaryotes, but still poorly
  understood

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Regulation of Enzyme Concentrations Degradation
(cont)

• Proteolytic degradation is an irreversible
  mechanism. For examples, rapid proteolytic
  degradation of enzymes that were activated in
  response to some stimulus (for example, in a
  signal transduction response). This type of
  down-regulation allows for a transient response
  to a stimulus instead of a continual response.
  Establishing the links between proteasomes,
  ubiquination and signal transduction pathways is
  currently a very active research area

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Zymogens Inactive Precursor Proteins

• A clinically important mechanism of controlling
  enzyme activity is the case of protease enzymes
  involved (predominantly) in food digestion and
  blood clotting. Protease enzymes (enzymes that
  degrade proteins) like pepsin, trypsin and
  chymotrypsin are synthesized first as larger,
  inactive precursor proteins termed zymogens
  (specifically pepsinogen, trypsinogen, and
  chymotrypsinogen, respectively).

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Zymogen Protease Examples
Chymotrypsinogen cleavage sites to yield active
chymotrypsin
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Zymogens (cont)

• Activation of zymogens by proteolytic cleavage
  result in irreversible activation. Zymogen forms
  allow proteins to be transported or stored in
  inactive forms that can be readily converted to
  active forms in response to some type of cellular
  signal. Thus they represent a mechanism whereby
  the levels of an enzyme/protein can be rapidly
  increased (post-translationally). Other examples
  of zymogens include proinsulin, procollagen and
  many blood clotting enzymes (the latter will be
  discussed in the next lecture).

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Enzyme/Substrate Compartmentation

• Segregation of metabolic processes into distinct
  subcellular locations like the cytosol or
  specialized organelles (nucleus, endoplasmic
  reticulum, Golgi apparatus, lysosomes,
  mitochondria, etc.) is another form of
  regulation. Enzymes associated with a given
  pathway frequently form organized,
  multi-component macromolecular complexes that
  perform a particular cellular process.
  Similarly, it follows that the substrates
  associated with a given pathway can also be
  localized to the same organelle or cytosolic
  location. This segregation allows for more
  specialized regulation of cellular processes.

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Enzyme Regulation by Compartmentation
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Allosteric Enzymes

• Allosteric enzymes - from the Greek allos for
  "other" and stereos for "shape" (or site) meaning
  "other site". These enzymes function through
  reversible, non-covalent binding of a regulatory
  metabolite at a site other than the catalytic,
  active site. When bound, these metabolites do
  not participate in catalysis directly, but lead
  to conformational changes in one part of an
  enzyme that then affect the overall conformation
  of the active site (causing an increase or
  decrease in activity, hence these metabolites are
  termed allosteric activators or allosteric
  inhibitors).

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Allosteric Example

• Feedback Inhibition - This occurs when an
  end-product of a pathway accumulates as the
  metabolic demand for it declines. This
  end-product in turn binds to the regulatory
  enzyme at the start of the pathway and decreases
  its activity - the greater the end-product levels
  the greater the inhibition of enzyme activity.
  This can either effect the Km or Vmax of the
  enzyme reaction.

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Metabolic Pathway Product/ Feedback Inhibition
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Allosteric Enzymes - Properties

• Allosteric enzymes differ from other enzymes in
  that they are generally larger in mass and are
  composed of multiple subunits containing active
  sites and regulatory molecule binding sites. The
  same principles that govern binding of a
  substrate to an active site are similar for an
  allosteric regulator molecule binding to its
  regulatory site.

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Kinetics of Allosteric Enzymes - Terms

• Cooperativity - in relation to multiple subunit
  enzymes, changes in the conformation of one
  subunit leads to conformational changes in
  adjacent subunits. These changes occur at the
  tertiary and quaternary levels of protein
  organization and can be caused by an allosteric
  regulator.
• Homotropic regulation - when binding of one
  molecule to a multi-subunit enzyme causes a
  conformational shift that affects the binding of
  the same molecule to another subunit of the
  enzyme.
• Heterotropic regulation - when binding of one
  molecule to a multi-subunit enzyme affects the
  binding of a different molecule to this enzyme
  (Note These terms are similar to those used for
  oxygen binding to hemoglobin)

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Allosteric Enzymes - Kinetics

• Allosteric enzymes do exhibit saturation kinetics
  at high S, but they have a characteristic
  sigmoidal saturation curve rather than hyperbolic
  curve when vo is plotted versus S (analogous to
  the oxygen saturation curves of myoglobin vs.
  hemoglobin). Addition of an allosteric activator
  () tends to shift the curve to a more hyperbolic
  profile (more like Michaelis-Menten curves),
  while an allosteric inhibitor (-) will result in
  more pronounced sigmoidal curves. The
  sigmoidicity is thought to result from the
  cooperativity of structural changes between
  enzyme subunits (again similar to oxygen binding
  to hemoglobin). NOTE A true Km cannot be
  determined for allosteric enzymes, so a
  comparative constant like S0.5 or K0.5 is used.

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Vo vs S for Allosteric Enzymes
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Models of Allosteric Proteins
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Regulation by Modulator Proteins - Calmodulin
Calmodulin is a small protein (17 kDa) that can
bind up to four calcium ions (blue dots) in the
two globular domains. When calciumis bound,
calmodulin acts as a protein co-factor to
stimulate the activity of target regulatory
kinases like phosphorylase kinase, myosin
kinase, Ca-ATPase and a Ca/calmodulin-dependent pr
otein kinase. It is the structural conformation
of Ca-calmodulin that makes it an active co-factor
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Regulation of Enzyme Activity by Covalent
Modifications

• Another common regulatory mechanism is the
  reversible covalent modification of an enzyme.
  Phosphorylation, whereby a phosphate is
  transferred from an activated donor (usually ATP)
  to an amino acid on the regulatory enyme, is the
  most common example of this type of regulation.
  Frequently this phosphorylation occurs in
  response to some stimulus (like a hormone or
  growth factor) that will either activate or
  inactivate target enzymes via changes in Km or
  kcat.

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Phosphorylation/Signal Transduction

• Phosphorylation of one enzyme can lead to
  phosphorylation of a different enzyme which in
  turn acts on another enzyme, and so on. An
  example of this type of phosphorylation cascade
  is the response of a cell to cyclic AMP and its
  effect on glycogen metabolism. Use of a
  phosphorylation cascade allows a cell to respond
  to a signal at the cell surface and transmit the
  effects of that signal to intracellular enzymes
  (usually within the cytosol and nucleus) that
  modify a cellular process. This process is
  generically referred to as being part of a signal
  transduction mechanism

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Signaling Regulation of Glycogen Synthase and
Phosphorylase
A-forms, most active B-forms, less active
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Other covalent modificiations

• Prenylation, Myristoylation, Palmitoylation The
  covalent addition of hydrophobic, acyl fatty acid
  or isoprenoid groups to soluble proteins/enzymes
  can alter their intracellular location. This type
  of hydrophobic acylation generally causes target
  proteins to associate with a membrane rather than
  the cytosol. Thus, it represents a mechanistic
  and functional re-compartmentalization of the
  target protein/enzyme (an example of a prenylated
  protein is the Ras oncogene discussed in lecture
  11)

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Allosteric and Phosphorylation Regulation -
Glycogen Phosphorylase