Spectrophotometric determination of a single pKa value

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• Spectrophotometric determination of a single pKa
  value
• Determination of a single pKa value of an
  acid/base pair with an oxidizable base
• Kinetic determination of a single pKa value
• Kinetic determination of two pKa values

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• Spectrophotometric determination of a single pKa
  value
• Biochemistry, 2003, 42, 14541-52, Yee et al.
• E. coli ribonucleotide reductase catalyzes the
  reduction of NDPs to dNDPs. The catalytic cycle
  is initiated by a radical, that is produced by a
  radical diferric-tyrosyl cofactor (in subunit R2)
  35 Å away from the active site (in subunit R1).
  The radical is transported by PCET (proton
  coupled electron transport). Which amino acids
  are involved?
• One candidate is Tyr356 because Phe356 protein is
  inactive. It was tested by replacement to
  difluorotyrosine and nitrotyrosine via chemical
  synthesis and intein technology.

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• Spectrophotometric determination of a single pKa
  value
• Difluorotyrosine (F2Y) has a pKa in the
  physiological range (7.8) but a redoxpotential
  similar to Tyr. The activity of an F2Y protein
  should be affected by pH because only the
  protonated form works for PCET. But does F2Y have
  the same pKa in solution as in the protein? To
  test this, Tyr is replaced by nitrotyrosine
  (NO2Y) because its properties can be measured by
  absorption.

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• Spectrophotometric determination of a single pKa
  value

PCET proton-coupled electron transfer RNR
ribonucleotide reductase
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• Spectrophotometric determination of a single pKa
  value

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• Chemistry

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• Goal pKa value
• Measurements pH dependence of the absorption
• Prediction pH dependence of the absorption
• Assumption absorption of the protonated and
  deprotonated state are additive, single
  protonation/deprotonation step,
  Henderson-Hasselbalch equation
• Unknown parameters pKa, absorption of the
  protonated and deprotonated state

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• Equations
• Henderson-Hasselbalch equation
• Conservation of mass
• Beer-Lambert law

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• A(0) absorption of NO2Y(0)
• A(1-) absorption of NO2Y(1-)
• A(pH) total absorption of NO2Y at a specific pH
• eNO2Y(0) absorption coefficient of NO2Y(0)
• eNO2Y(1-) absorption coefficient of NO2Y(1-)

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• Eliminate A(0) and A(1-)
• Eliminate NO2Y(0)
• Eliminate NO2Y(1-)

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• Introduce A0 and A1-
• A0 absorption, if all NO2Y is protonated
• A1- absorption, if all NO2Y is deprotonated
• and eliminate NO2Yges, eNO2Y(0) and eNO2Y(1-)

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pKa 7.0, 8.0
pKa 7.05, 7.96
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• Result The pKa of NO2Y (7.0) is considerably
  lower than that of Tyr (10.5). Comparison of the
  pKas of peptide, subunit R2, and complexes with
  R1 and substrates shows little perturbation of
  the pKa (differences lt 1) by the protein
  environment. This behavior can be transferred to
  Tyr and other Tyr derivatives, for example
  2,3-difluorotyrosine. Here, the redox potential
  is similar to Tyr (in contrast to NO2Y, whos
  redox potential is higher than for Tyr), but the
  pKa cannot be directly measured. Enzyme activity
  is very low for the NO2Y-derivative.

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Same pH profile
F2Y has a lower pKa, but it shows the same pH
dependence as wild type (with Tyr). Thus, radical
initiation (transport from R2 to R1) seems not
to use Tyr356 in a proton-coupled electron
transport.
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• The redox potential can also be used to determine
  the pKa because the protonated phenol is much
  harder to oxidize (a proton and/or the nitro
  group make it harder to remove an electron).

Nitrotyrosine
Tyrosine
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The cationic radical is unstable. Therefore,
pKa,ox is low and E0,acidic is high.
In the following, only pKa,red and E0, bas are
considered.
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• Goal pKa value
• Measurements pH dependence of the redox
  potential
• Prediction pH dependence of the deprotonated
  reduced and oxidized state
• Assumption single protonation/deprotonation step
  with oxidation of the deprotonated form (the
  protonated oxidized form (cation radical) is
  considered unstable), Henderson-Hasselbalch
  equation, Nernst equation
• Unknown parameters pKa,red, E0,bas at high pH

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• Equations
• Henderson-Hasselbalch equation
• Conservation of mass (reduced forms)
• Nernst equation

(1)


(2)
(3)
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• NO2Y(0) protonated nitrotyrosine
• NO2Y(1-) deprotonated nitrotyrosine
• NO2Y(0) deprotonated oxidized nitrotyrosine
  radical
• NO2Yred concentration of all reduced species
  (protonated and deprotonated form)
• E redox potential of the nitrotyrosyl
  radical/nitrotyrosinate pair
• E0 standard redox potential of E

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• At low pH NO2Y(1-) is decreased compared to
  high pH (NO2Yred) (eliminate NO2Y(0), 2 in 1)
• (4)
• NO2Y(0) is assumed pH independent, but red
  (i.e. NO2Y(1-)) is pH dependent (see 4). E can
  then be described with a pH dependent E0.
  Reference is E0(high pH), where all reduced
  nitrotyrosine is deprotonated.

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Tyr / NO2Y pKa - / 7.0 E0
- / 1.02 V
Tyr / NO2Y pKa 9.85 / 6.98 E0
0.64 V / 1.00 V
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• Kinetic determination of a single pKa value
• Biochemistry, 2003, 42, 15179-88, Ghanem et
  al.

Ein ander mal
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• Kinetic determination of two pKa values
• Biochemistry, 2003, 42, 14614-25, Stefanova
  et al.
• In the example the fast acylation of the
  penicillin binding protein 3 (PBP 3) from
  Neisseria gonorrhoeae is tested for its pH
  dependence. PBPs are characterized and detected
  by their binding of the antibiotic penicillin.
  The physiological role of the PBPs is within the
  synthesis of the cell wall peptidoglycan that
  surrounds the bacteria.
• Transpeptidase activity of the PBPs

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• Peptidoglycan

NH2
COOH
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• Crosslinking reaction

2. The amino group is attached to the D-Ala.
NH2
1. The C-terminal D-Ala is cleaved off and the
PBP is attached to the second last D-Ala.
COOH
COOH
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• For substrate binding (at the D-Ala-D-Ala part of
  the peptidoglycan) and the mechanism of
  transpeptidation (acylation and deacylation) the
  protonation state of the enzyme is important.
• Penicillin inhibits the transpeptidation, because
  it acylates the protein fast, but only very
  slowly deacylates (--gt penicillin binding
  protein).
• The reaction with penicillin is useful for the
  study of the acylation reaction.

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• The reaction with penicillin can be described by
  binding, acylation and deacylation
• k2/K describes formation of the acyl enzyme at
  low concentrations of b-lactam antibiotic
  (penicillin).
• K (k-1 k2)/k1.

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• Goal pKa values
• Measurements pH dependence of the k2/K
• Prediction pH dependence of the reaction (k2/K)
• Assumption two protonation/deprotonation steps,
  two Henderson-Hasselbalch equations, only the
  singly protonated species is active
• Unknown parameters pKa,1, pKa,2, k2/K for the
  singly protonated species

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• The two deprotonation steps
• The equilibria and Henderson-Hasselbalch
  equations
• (1)
• (2)

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• The enzyme must be in one of the three
  protonation states
• (3)
• Each species could contribute to the acylation
  rate
• (4)

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• Eliminate EH2 (3 and 1a) and calculate EH
• (5)
• Eliminate EH (5 in 2a)
• (6)

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• Use (6) to calculate EH with (2a)
• (7)
• Use (7) to calculate EH2 with (1a)
• (8)

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• Now we asume, that the low pH and the high pH
  species are inactive ( Acyllow Acylhigh 0)
• (9)
• This is the same form as used in the paper

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• The separately calculated values are not very
  different from the overall calculation, but at
  least the number of parameters in the second case
  is lower. This is preferred.