Enzymecatalytic reaction is the new alternative plan

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Introduction

• Enzyme-catalytic reaction is the new alternative
  plan
• that can substitute the present industrial
  chemical process.
• The main drawback of it may the low synthetic
  yield of the product.
• The use of organic cosolvent systems may improve
  the yield
• by increasing the solubility of substrates,
  reducing the water activity.
• Previous research, the effects are tested in the
  enzymatic synthesis of pivampicillin(PVM)
• from D-?-phenylglycine methyl ester(PGM) and
  pivaloyloxymethyl 6-aminopenicillanic
• acid(POM-6-APA) by using penicillin acylase
  catalyst.

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Introduction
1 D-?-phenylglycine methyl ester(PGM) 2
POM-6-APA (RCH2OCOC(CH3)3) 3
pivampicillin(PVM) (RCH2OCOC(CH3)3) 4
D-?-phenylglycine Fig. Reaction mechanism for
penicillin acylase-catalyzed synthesis of PVM.
? But in fact both reaction yield and rate are
all reduced by small amount of organic
cosolvent. (see table1)
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Introduction
Table. The reduced experimental reaction
rate(v/v0) by organic cosolvents at pH6.0,
T295.15?C.
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Problems and its solutions

• Find main cause which change the reactivity of
  Penicillin acylase.
• ? Partial charge variation of penicillin acylase
  in organic cosolvents
• How can we theoretically prove the inhibition
  effects?
• ? Potential field analysis by BEM and the
  Brownian Dynamics
• Is this theory well-correlated with experimental
  results?

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3D structure of Penicillin acylase
Fig. The key residues which bind the substrate
are located at the back of the active site.
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Partial charges of Penicillin acylase

• By Handerson-Hasselbalch equation

• If we know a bulk pH values(given) and pK values
  of molecules, we can assign partial charge.
• ( NZ with tyr, SG with cys, OG with ser and thr,
  OH with lys,
• between OD1 and OD2 with asp, between OE1 and
  OE2 with glu,
• between NE1 and NE2 with his, between NH1 and
  NH2 with arg )
• We already know pK in aqueous solution, but
  dont know in organic solvents mixture
• Therefore we must find pK value of protein
  residues in organic cosolvents

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Partial charges of Penicillin acylase

• Charge value

• Charge position

Fig. Partial charge position in protein residues.
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pK value in organic solvents

• From Nernst equation

where DG is the solvation free energy, pH is bulk
phase and given, R is ideal gas
constant, T is temperature.
? The hydration free energy is the energy of
transferring a molecule from the gas phase
into an infinite bath of water.
? Similarly, the solvation free energy is the
energy of transferring a molecule from the
gas phase into an infinite bath of solution.
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pK value in organic solvents
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pK value in organic solvents

• By numerical computation (BEM)

Fig. Comparison calculated and theoretical pKa
value. Experimental(?), calculated(?). Errors
() arg(3.39) asp(11.7), cys(9.72), glu(19.32),
his(5.38), lys(5.81), Ser(0.15), thr(17.28),
tyr(2.89).
Table. Hydration energy(kcal/mol) and calculated
pKa values.
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pK value in organic solvents
Fig. pKa value variation in pure organic
solvents(left) and 2 mixture(right) according to
solvents e. arg(?), asp(?), cys(?), glu(?),
his(?), lys(?), ser(?), thr(?), tyr(?).
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Potential field analysis

• Now, we have the partial charge data of
  penicillin acylase.

• First, we qualitatively test the reactivity
• by potential field around the penicillin
  acylase by BEM,

• Applying Greens second identity to governing
  equations,

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Potential field analysis
Fig. Surface discretization of PA by Connollys
MSP (7762 triangular elements and 15520 nodes)
For actual calculation, we apply some idea
which reduce elements number.
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Potential field analysis
Fig. Potential field of PA at pH6.0 and
k9.6-1(left) and k0.0(right) in unit kT/e.
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Potential field analysis
Fig. The potential field(rectangle of fig.7) of
PA in water(left) and in glycerol 2
mixture(right) well-reacted(102v/v0)
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Potential field analysis
Fig. The potential field (rectangle of fig.7) of
PA in ethyl acetate 2 mixture(left) 31v/v0
and in tetrahydrofuran 2 mixture(right)
almost 0.0v/v0 .
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Simple properties of PA
Fig. The correlation between simple properties of
PA and experimental reactivity. (hydrophobicity
upper left, solvent dielectric constant upper
right, net charge near active site lower left,
dipole moments near active site lower right)
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The Brownian Dynamics

• Fail to quantitatively explain the correlation
• between experimental and calculated reactivity
  data.
• Another two quantity, the binding energy and the
  rate constant
• are calculated by mean of Brownian Dynamics.
• Structure data(pdb file) is in web
  site(http//www.rcsb.org/pdb),
• and charges and radii parameters are already
  calculated.
• For convenience, the calculation of two
  properties carried out by using UHBD.
• ? Further study about Molecular
  Dynamics is needed.

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The Brownian Dynamics
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The Brownian Dynamics
? The binding energy
Fig. The binding energy calculation results.
There is no correlation between experimental
reactivity and the energy(left). But the total
charge of penicillin acylase is good related with
the energy(right). Linear regression result is
y9.983.74x, r20.91.
? It is caused by the binding energy is based
only on the charge distribution. Another reaction
effects can not be applied.
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The Brownian Dynamics
? The diffusion controlled rate constant
Fig. The diffusion controlled rate constant
results. There is good correlation between
experimental and calculated rate constant ratio
to case of aqueous solution. Data which are in
red ellipse are the mixture of ring structured
and acidic cosolvents, e.g., cyclohexanone,
dioxane, tetrahydrofuran, pyridine and
phenol(ring structured) and isobutylic
acid(acidic). These have abnormally-low reaction
rate. Its causes are not yet revealed.
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Conclusions

• Key points for reasonable explanation of the
  inhibition effects of organic cosolvents

• Shift of pKa value of amino acid due to organic
  cosolvents
• Partial charge variation due to the pKa shift
• Positive potential channel around the active
  site of PA
• Rate constant calculation by Brownian Dynamics

• From this research, we can predict the
  reactivity of enzyme in organic cosolvents.
• This method can be extended to not only
  penicillin acylase but also other enzyme and
• general proteins. Moreover, a property which
  varies in organic cosolvents can be well
• explained in the future.