Proteins and Enzymes

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Proteins and Enzymes BIOL 239 BMSC 209 Enzyme
Function Paul Teesdale-Spittle KK712, Ext 6094,
e-mail paul.teesdale-spittle_at_vuw.ac.nz
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• The catalytic power and specificity of an enzyme
  is a product of the protein structure.
• structural complimentarity with the substrate
• the ability to generate localised chemical
  environments around substrate.
•  
• How can reaction rates be increased?

Where k is the rate constant, X referes to the
concentration of X, A relates to efficiency and
orientation effects, EA is the activation energy,
R is the gas constant and T is the temperature
(in K).
Rate k.X.Y
(Arrhenius equation)
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Brainstorm ways of increasing reaction rates
Define these as suitable or unsuitable for use
in a cellular environment.
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What do proteins do to catalyse reactions?

• Acid base catalysis
• Electrostatic interactions
• Covalent catalysis (intermediate formation)
• Proximity and orientation effects
• Strain
• Changes in reaction conditions.

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• The overall effect of an enzyme is to lower the
  free energy of activation (EA) for the reaction
• by providing an alternative reaction pathway
• by reducing the energy gap between substrates
  and transition state
• stabilising the transition state of the usual
  pathway
• binding the substrate in a high energy
  (strained) conformation
• by binding two or more reactants in the
  orientation required for reaction to occur
• Increasing the effective local concentration.

Consider the rate and Arrhenius equation again
Rate k.X.Y
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• The next sections will look at a few enzymes and
  identify which of the catalytic strategies are
  being used in each case.
• Each enzyme will be considered under the
  following headings
• Protein 
• Function
• Underlying chemistry
• Catalytic strategy
• Mechanism and structural features

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• Lysozyme
• Hen egg white lysozyme (HEWL) 14.6 kD, 129 aas.
• Function
• Destruction of bacterial (and fungi) cell walls.
• Hydrolysis of glycosidic linkages.
• HEWL rate 108 faster than non-catalysed
• Found widely in vertebrates
• Possible role in bacteriocidal action.
• More likely role in cleaning away dead
  bacteria.

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• Underlying chemistry
• Gram-positive bacteria have crosslinked
  peptidoglycan cell walls.
• The oligosaccharide is an alternating
  arrangement of two saccharide subunits
• 2-acetamido-2-deoxyglucopyranoside (NAG)
• 2-acetamido-2-deoxy-3-O-lactylglucopyranoside
  (NAM)

NAG (notice glucose with 2-OH replaced with
NHAc.
NAM (notice NAG with lactate on 3-O.
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NAG and NAM are ?(1?4)-linked. Many fungi cell
walls contain chitin, which is ?(1?4)-linked
poly(NAG).
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So the chemistry is the cleavage of an ether bond
of an acetal
Chemically this reaction results from treatment
with a dilute mineral acid, with the following
mechanism
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• Catalytic strategy
•  HEWL uses two key residues in the catalytic
  mechanism
• Glu 35 acid and base catalysis,
  proximity/orientation of hydrolysing water
  molecule.
• Asp 52 covalent catalysis
• Mechanism and structure
• Two previously proposed mechanisms.
• Recent crystallographic results (Vocaldo et al,
  Nature, 2000, 412 (23rd August issue), 835-838)
  demonstrate mechanism discussed here.
• This is different than the mechanism in most
  text books!

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N.B. highly simplified polysaccharide!

• Which?
• Acid base catalysis
• Electrostatic interactions
• Covalent catalysis (intermediate formation)
• Proximity and orientation effects
• Strain
• Changes in reaction conditions.

Saccharide
H2O
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How was the mechanism solved?

• In native enzyme with native substrate k3gtgtk2
• To trap E-S covalent intermediate this must be
  reversed
• k3 reduced whilst k2 maintained.
• Converted Glu 35 to Gln 35, so no base catalysis
  in k3 step.
• Used activated S so no affect of loss of acid
  catalysis in k2 step.
• Detected covalent E-S intermediate by MS.
• Could not detect by crystallography as turnover
  still too fast.
• Modified S by inclusion of EWG in place of
  N-acetyl group.
• Slowed k3 step (WHY?) and allowed
  crystallography.

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• RNase A
• 124 aas
• 4 S-S bonds
• Can be cleaved by 2-mercaptoethanol and 8M urea.
• Oxidatively renaturation after removal of urea
  (O2, pH 8) occurs with retention of activity.
• Oxidative renaturation in presence of 8M urea
  leads to 1 of activity regained.
• The inactive oxidised form can be made active by
  treatment with a low concentration of 2
  -mercaptoethanol
• Discuss - WHY??
• Good example of thermodynamically driven folding
  specificity.

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• Protein Disulphide Isomerase (PDI) enzyme
  catalyses the disulphide bond formation.
• Function
• There are many different RNases.
• They hydrolyse RNA to constituent nucleotides
• Here concentrating on RNase A
• Underlying chemistry
• Breaking 5-O to phosphate ester bond in RNA
• Proceeds via a 2,3-cyclic nucleotide
  intermediate, which is then hydrolysed.

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• Catalytic strategy
• RNase A uses two catalytic His residues, which
  act both as acids and bases during the catalytic
  cycle.
• His 12
• acts as a base to activate the 2-OH group to
  aid formation of the cyclic phosphate ester
  intermediate
• acts as an acid to assist opening of the cyclic
  phosphate ester intermediate
• His 119
• acts as an acid to assist cleavage of the 5-O to
  P bond
• acts as a base to activate water to hydrolyse
  the cyclic phosphate ester intermediate

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Mechanism and structural features

• Which?
• Acid base catalysis
• Electrostatic interactions
• Covalent catalysis (intermediate formation)
• Proximity and orientation effects
• Strain
• Changes in reaction conditions.

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His 12
His 119
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• Kinesin
• Kinesins (along with dyneins) are responsible
  for transport of vesicles and organelles within
  cells.
• Uses microtubules as a track
• Transport away from the cell centre (dyneins and
  some other kinesin-related proteins transport in
  opposite direction)
• Powered by ATP.
• Have several domains
• The "head" or motor domain (containing the
  microtubule and ATP binding sites)
• Globular binds microtubule ATP and generates
  the motive force.
• A "stalk," which is largely ?-helical.
• A "tail" connecting the stalk to the cargo.

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• Focus on the motor domain.
• 330 residues
• Central sheet of eight ?- strands sandwiched
  between three ?-helices on either side.
• There are also numerous loops.

Neck builds from here
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Models of tubulin nucleation. E. Nogales.
Structural Insights Into Microtubule Function.
2000. Annu. Rev. Biochem. 69, 277-302.
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• Full movement along tubulin occurs with dimeric
  kinesin
• Some movement with monomeric kinesin.
• Model of movement is hand over hand processive
  motion.
• the trailing head detaches and rebinds to the
  next open tubulin dimer site on the same tubulin
  filament
• generates the 8 nm step size.
• Movement is through conformational adjustments
  as ATP hydrolyses to ADP.
• The kinesin-ADP complex is long lived
• Believed to associate weakly with tubulin.
• Kinesin-ATP complex forms transiently (never
  been isolated).
• Believed to bind strongly to tubulin.

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• The neck linker has to be released from its
  interaction with the core to allow this motion to
  occur.
• The nucleotide of kinesin is embedded in four
  contact regions (N-1 to N-4).
• Homologues in a number of other proteins.
• Some of these contact region homologues are
  known to act as conformational switches,
  generating movement of protein domains relative
  to each other.
• N-1 ?,?-phosphate GQTxxGKS/T 8693
• N-2 Switch-I ? -phosphate NxxSSR 199204
• N-3 Switch-II ? -phosphate DxxGxE 232237
• N-4 base RxRP 1417

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• Notes
• Water relays some interactions
• Mg
• ? stacking
• ADP, so no ?-phosphate

N-1 ?,?-phosphate 8693 N-2 Switch-I ? -phosphate
199204 N-3 Switch-II ?-phosphate 232237 N-4
base 1417
Remember Koshland and put in ATP protein moves!
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• Summary of proposed model
• ATP binds. Kinesin as a dimer binds strongly to
  tubulin.
• The trailing head hydrolyses ATP to ADP (THE
  CATALYTIC EVENT!!). This can dissociate from
  tubulin.
• Movement of the protein is relayed via
  switches in the nucleotide binding region
  (possibly 199204 and 232237).
• The movement is facilitated by flexibility in
  the neck region.
• The newly freed ADP-kinesin swings to the next
  free tubulin dimer binding site, 8 nm away.
• Rebinding to tubulin facilitated by ADP to ATP
  exchange.
• The new trailing head starts the cycle all over
  again
• (S. Sack, F.J. Kull and E. Mandelkow. 1999. Motor
  proteins of the kinesin family. Structures,
  variations, and nucleotide binding sites. Eur.
  J. Biochem. 262, 1-11
• http//www.ejbiochem.org/cgi/content/full/262/1/1
  ).

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• The motor domain interacts mainly with
  ?-tubulin.
• Stoichiometry of one head per tubulin
  heterodimer
• The step size of movement is 8 nm, equivalent
  to the axial spacing of tubulin heterodimers.

The three- dimensional map of microtubules is
shown in surface rendering (green) The heads of
attached kinesin dimers are shown in carbon
backbone representation (yellow). The neck helix
is red, The region beyond the neck helix is
shown schematically as a red chain.
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Surface rendered image reconstructions of
microtubules decorated with a monomeric kinesin
construct (called rK354). Tubulin subunits are
blue. (Note saturation of binding sites required
by imaging technique, not necesarilly the
situation in cells.
A. Hoenger, S. Sack, M. Thormählen, A. Marx,
J. Müller, H. Gross, and E. Mandelkow (1998).
Image Reconstructions of Microtubules Decorated
with Monomeric and Dimeric Kinesins Comparison
with X-Ray Structure and Implications for
Motility. J. Cell Biol., 141(2),,
419-430http//www.jcb.org/cgi/content/full/141/2/
419
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R. Stracke, K.J. Bohm, J. Burgold, H.J. Schacht,
E. Unger. 2000. Physical and technical
parameters determining the functioning of a
kinesin-based cell-free motor system.
Nanotechnology, 11 (2), 52-56 Kinesin is a
microtubule-associated protein, converting
chemical into mechanical energy. Based on its
ability to also work outside cells, it has
recently been shown that this biological
machinery might be usable for nanotechnological
developments. .. This paper reports on the
example of microtubules gliding across
kinesin-coated surfaces .. Individual
microtubules were observed to cover distances of
at least 1 mm without being detached from the
surface and to overcome steps of up to 286 nm
height. In addition, mechanically induced how
fields were shown to force gliding microtubules
to move in one and the same direction. This
result is regarded as being an essential step
towards future developments of kinesin-based
microdevices as this approach avoids
neutralization of single forces acting in
opposite directions.
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Must do carboxypeptidase A chymotrypsin and
serine proteases