Enzymologie moléculaire et...

Université de Montréal

Faculté de Médecine Département de biochimie



Enzymologie moléculaire et mécanistique

Experimental approaches in enzymology

Part 1





Proton transfer

• Proton transfer between chemical groups is one of the fundamental processes catalyzed by enzymes

• Proton transfer can be part of more complex overall reaction, provide a route for changing bonding and geometry, as well as redistributing charge in a substrate molecule

• Energetics of proton transfer is determined primarily by the pKa`s of donor and acceptor groups

• Simplest mechanism for enzymes to catalyze proton transfer is by changing the relevant pKas

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Rules of hydrogen bonding

1: The greater the charges, the stronger the hydrogen bond.

2: The shorter the distance, the stronger the hydrogen bond.

– Hydrogen bond length is traditionally measured by the distance between the donor atom and the acceptor atom.

– Hydrogen bond occurs if the distance between the donor and the acceptor atoms is shorter than the sum of the atomic radius of the acceptor atom (~1.5Å), the atomic radius of the hydrogen (1.2Å) and bond length between the donor atom and the hydrogen (~1Å). So the longest hydrogen bonds are ~3.5 Å. Anything longer would be considered a pure dipole-dipole interaction.

– Good hydrogen bonds have a distance of ~2.8 Å and ultra-short hydrogen bonds have been reported with donor to acceptor distances of 2Å.

Partial charge separation induceselectrostatic dipole-dipole interaction; 1/r3 dependence – interaction onlyimportant up to nearest neighbours

Hydrogen bonds are formed when an electronegative atom approaches a hydrogen atombound to another electronegative atom.

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3: Bonding angles matter, the more ideal the bonding angle, the stronger the hydrogen bond.

– The bond is ideal if the donor atom, the hydrogen atom, the lone pair and the acceptor atom all lie on a straight line. Angular dependence implies that hydrogen bonds have a partial covalent character.


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• Quantum mechanical simulation of the bonding strength between two water molecules show that:

– The interaction energy is favourable if the free electron pairs of the acceptor molecule are pointing towards the donor but become equally unfavourable otherwise.

Energy (kcal mol-1)





4: Linear Networks of hydrogen bonds increase the dipole moment and lead to stronger hydrogen bonds.

– The dipoles in hydrogen bonds are induced dipoles. Formation of a hydrogen bond further polarized the bonds.

5: Hydrogen bonds contribute little to overall protein stability, but they align molecular groups in a specific orientation giving proteins a defined structure.


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LBHB (Low Barrier Hydrogen Bond)

LBHBs are stronger than weak hydrogen bonds; weak hydrogen bonds strength depends on the type of heteroatoms, their electrostatic charge, the distance separating them, and the dielectric medium.

LBHB => matching pKa’s

2–8 kcal mol-1 10–20 kcal mol-1 24–40 kcal mol-1

Cleland WW. Low-barrier hydrogen bonds and enzymatic catalysis. Arch Biochem Biophys. 2000 Oct 1;382(1):1-5. Review.

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LBHB: Physicochemical Properties

• BOND LENGTHS

– Bond distance < 2.6Å for O· · ·O, < 2.7Å for O· · ·N, and < 2.8Å for N· · ·N.

• SPECTROSCOPIC PROPERTIES

– NMR signal for an LBHB involving O or N is downfield in the range of 18–20 ppm on the chemical shift scale

– NMR chemical shift of the proton ranges typically between 9 and 11 ppm but may be higher up to ~ 15ppm

• DEUTERIUM FRACTIONATION FACTORS : fractionation factor is the equilibrium constant for the exchange of hydrogen bonded protons with deuterons in deuterium oxide.

– In an LBHB, hydrogen is more strongly bonded than deuterium because of the effect illustrated in Figure 1B (entropic effect). Deuterium, feels less attraction to the second heteroatom, owing to its lower zero point energy.

• The deuterium fractionation factor for an LBHB is therefore < 1.0, typically 0.3–0.7

• Weakly hydrogen bonded protons display deuterium fractionation factors ~ 1.0–1.2

– Measured by NMR

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pKa and microenvironment

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Protein Ionizable Groups: pK Values

Pace CN et al. J. Biol. Chem. 2009;284:13285-13289

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Factors influencing the pK values of ionizable groups in proteins

Pace CN et al. J. Biol. Chem. 2009;284:13285-13289

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Dehydration – Born effect

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Charges in the hydrophobic interior of proteins

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GdnHCl denaturation

monitored by Trp

fluorescence

G° is a function of [GdnHCl]

Changes of emission spectra (excited at 280 nm) at various concentration of GdnHCl.

GdnHCl

0 M 1 M2 M4 M 6 M

Jana S et al. Effects of guanidine hydrochloride on the conformation

and enzyme activity of streptomycin adenylyl-transferase monitored

by circular dichroism and fluorescence spectroscopy. Biochemistry

(Mosc). 2006 Nov;71(11):1230-7.

SiF signal of folded state

SiU signal of unfolded state

SiO observed signal at a given

denaturant concentration

Fraction of unfolded structural element

ΔGi - difference in free energy between the folded and the unfolded states at the given concentration of denaturant, itype of experiment, mi is a measure of the dependence of ΔGi on denaturant concentration [D], ΔG0

i (H2O) measures intrinsic stability in water



Measurement of pKa values of internal ionizable groups by analysis of pH dependence of thermodynamic stability in Staphylococcal Nuclease

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pKaN and pKaD refer to the pKa of residue in the native and in the denatured states, respectively. The pH-dependent contributions to ΔΔGH2O (pH) are captured by the right-most term. pH-independent component (ΔΔGH2O (mut)) reflects the energetic consequences of the mutation independent of the electrostatic effects associated with shifts in pKa. Function assumes that the pH dependence of ΔΔGH2O

is determined solely by the pKa of the residue and assumes that the mutation does not significantly affect the ionization properties of other titratable groups.

Isom DG et al. PNAS 2010;107:16096-16100

native

denatured

WT

L25E





Isom DG et al. PNAS 2010;107:16096-16100

rcav = 2 Årprot = 12 Å

pKa values of Glu residues in 25 internal positions in Staphylococcal Nuclease

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pKa Dapp

ΔG (kcal mole-1)

V104E 9.4 9.2 6.7

L37E 5.2 38.1 1.0

H2O 78.5

Methane 2.8

L25EL37E

V104E





• Blue lines identify cases where predenaturational transitions suggest partial structural changes coupled to the ionization of the internal Glu.

• Red lines identify the case where the titration of the internal group coincides with the major unfolding transition.

Controls: Consequences of the Ionization of Internal Glutamate Residues

Trp fluorescence and far-UV circular dichroism as a

function of pH was used to monitor the effects of

internal charges on conformation.

• Tryptophan has a wavelength of maximum

absorption of 280 nm and an emission peak

that is solvatochromic, ranging from ~ 300 -

350 nm depending in the polarity of the local

environment.

• Approximate secondary-structure content of a

protein (‘protein has 40% α-helix and 20% β-

sheet’) can be estimated spectroscopically in

the far-ultraviolet (far-UV, 170–250 nm) by

circular dichroism

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Location of Glu residues that promote partial unfolding (blue), global unfolding (red), or no conformational reorganization coupled to ionization (gray)

Conformational Consequences of the Ionization of Internal Glu Residues

Data demonstrate that • Several charged glutamates buried in

proteins are destabilizing • Most charged Glu side chains can be buried

readily in the hydrophobic core of stable proteins o without need for specialized structural

adaptations to stabilize them o without inducing any major

conformational reorganization

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Two of 25 Glu residues titrated with normal pKa near 4.5

Other 23 titrated with elevated pKa values ranging from 5.2–9.4, with an average value of 7.7





Microenvironmental effect on pKa by charge-charge interactions

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Charge-charge interaction

• Charge-charge interactions are the chief perturbation of the pK values of ionizable groups on the protein surface

– where the majority of the ionizable groups are located in proteins

• Large shifts in pKa can be produced by spatially proximal cationic or anionic residues

– charge destabilization (Weistheimer’s hypothesis)

• When opposite charges are 4.2 Å apart in water,

– ΔG = ~ 1 kcal/mol,

– ΔG reduced to 0.5 kcal/mol at the ionic strength inside a cell

ΔGij = qi qj / D rij

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Charge-Dipole Interactions (Hydrogen Bonds)

RNase T1PDB entry 1RGG

pKa ~ 0.6

Thurlkill RL, Grimsley GR, Scholtz JM, Pace CN. Hydrogen bonding markedly reduces the pK of buried carboxyl groups in proteins. J Mol Biol. 2006 Sep 22;362(3):594-604.

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Microenvironmental effect of Hydrogen Bonds

• Ionisable groups can also interact with the partial charges or dipoles on neighbouring polar groups

– they can raise or lower the pK values by several pK units

• Example: The side chain carboxyl of Asp-76 in RNase T1 has a very low pKof 0.6 and forms three intramolecular hydrogen bonds to the side chains of Asn-9, Tyr-11, and Thr-91

– To see if these hydrogen bonds were responsible for the low pK, the hydrogen bonds were removed one at a time, and the pK of Asp-76 was measured.

• When single hydrogen bonds are removed, the average pK increases to 3.3.

• When two hydrogen bonds are removed, the average pK increases to 5.1.

• When all three hydrogen bonds are removed, the pK increases to 6.4.

– Thus, in the absence of the hydrogen bonds, the pK is elevated 2.5 units above the pKintrinsic. This increase results from the Born effect because the carboxyl group of Asp-76 is buried.

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Base Catalysis in Hydroxynitrile Lyase

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Base Catalysis in Hydroxynitrile Lyase• Hydroxynitrile lyase (HNL) (EC 4.1.2.39) catalyzes cleavage of cyanohydrins to

hydrocyanic acid plus the corresponding aldehyde or ketone. The release of HCN serves as a defense against herbivores and microbial attack for a variety of plants.

• Mechanism: Hydroxynitrile lyase uses a catalytic triad consisting of Ser80-His235-Asp207 to enhance the basicity of Ser80-O gamma for abstracting a proton from the OH group of the substrate cyanohydrin.

Stranzl GR, Gruber K, Steinkellner G, Zangger K, Schwab H, Kratky C. Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate. J Biol Chem. 2004 Jan 30; 279(5): 3699-707.

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Catalytic features• Key mechanistic elements are (for the cyanohydrin cleavage direction):

– deprotonation of the OH-Ser80 by His235 and concomitant abstraction of a proton from the substrate hydroxyl by Ser80.

– cleavage of the cyanohydrin is facilitated by the stabilization of the charge of the nascent cyanide through interaction with the positive charge of Lys236

• Hevea brasiliensis (Hb) HNL structure was refined against diffraction data collected to very high (1.1 Å) resolution.

– refinement yielded short distance between residues of catalytic triad: Nδ(His235) and Oϵ(Asp207) 2.67 Å

– Difference density indicated a proton attached to Nδ(His235), with no apparent density near Nϵ(His235) or Oϵ(Asp207). • Proton between His235 and Asp207 not shared among the two heteroatom.

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LBHB (SSHB – Short Strong Hydrogen Bond)

• Occurrence of SSHBs in considerable number of enzymes:

– ketosteroid isomerase, triosephosphate isomerase, serine proteases, tryptophan synthase, 2-amino-3-ketobutyrate-CoA ligase, cholinesterases

• Detected by NMR spectroscopy

– exposure of the delocalized proton decreases the electron density around the proton nucleus, which shifts the NMR signal to very low field (higher frequency; typically 18-22 ppm)

– fractionation factor ϕ for the associated proton, defined as the equilibrium constant for the exchange of hydrogen by deuterium with the solvent. • ϕ depends on the relative strength of a hydrogen bond compared with the

solvent, with values lower than 1 indicating stronger hydrogen bonds

• NMR-observable quantity





NMR Signal Assignment (free HbHNL)

empirical correlation between proton chemical shifts and hydrogen bond lengths in imidazolium·carboxylate complexes

D =1.99 + 0.198 ln(δ) + (10.14/δ)5 in Å δ = 15.41 ppm2.65 Å,

crystallographically observed distance is 2.67 Å

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+ 6 mM benzaldehyde;- ring current effect -

Fig. Full one-dimensional 1H-NMR spectrum of 0.63 mMsolution of HbHNL in 50 mM phosphate buffer, pH 7.4, at 298 K Fig. Two-dimensional 1H-15N-HSQC of a solution

containing 0.33 mM 15N-labeled HbHNL

N-H signal of Nδ1 of His235





Modelling - Control

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Fig A. Stereo diagram showing amodel of benzaldehyde (pink) in the active side of HbHNL. The center of the aromatic ring of benzaldehyde is 7.1 Å away from H-Nδ1(His235), and the line connecting this center with H-Nδ1(His235) is roughly perpendicular to the plane of the aromatic ring.

Fig B. The active site of HbHNL with a superposition of a molecule of acetone (violet) and thiocyanate (magenta) as observed in the respective complex crystal structures. Note that the water molecule labeled Wat of the HbHNL·acetone complex superimposes with the thiocyanate-nitrogen atom of the HbHNL·thiocyanate complex





One-dimensional 1H-NMR spectra of a 0.63 mM solution of HbHNL with various concentrations of thiocyanate (SCN-) inhibitor

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More characterization• Hydrogen-Deuterium Fractionation Factors

– 0.98 is obtained for HbHNL in the absence of thiocyanate– 0.35 in the presence of 1 mm thiocyanate.

• value typical for a short, strong hydrogen bond (LBHB)

• pH Titration– protonation properties of His235 investigated – pH titrations in the range between pH 4 and 10 for HbHNL in the

presence and in the absence of SCN-

• Between ∼pH 4 and 9, titration of free HbHNL did not lead to detectable spectral changes.

• In the presence of thiocyanate1. low pH side, no pH-dependent spectral changes are observed 2. the signal at 19.35 ppm starts to diminish above pH 7 with concomitant

reappearance of the 15.41 ppm signal in the presence of thiocyanate. 3. No further change at pH > 9.

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Need to understand stability of protonation states at His235



Protonation states for His – Asp diad

• Relative free energies of these four protonation states of the His235-Asp207 diadwere estimated as a function of pH

• Calculations used HbHNL structure as observed crystallographically – in the absence and

– in the presence of thiocyanate.

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Calculation for ΔG-values of His – Asp diads

• Calculation of ΔG-values of diadsbased on– pKa values of histidine and aspartic

acid in solution plus

– two parameters that are accessible by FDPB methods

1. the electrostatic interaction energy of the two residues in the hydrogen-bonded pair

2. the solvation free energy of the diad for the transfer from water into the protein environment.

Thermodynamic cycle

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Finite Difference Poisson-Boltzmann FDPB calculations (DelPhi program)

Each thermodynamic cycle starts from the two residues in water at infinite separation, where the relative free energies ΔG0 depend only on the pKa

values of the two residues (6.5 for His and 4.4 for Asp) and on the solution pH for the transitions from zwitterionic to cationic or anionic state.

The approach of the two solvated groups results in the formation of different types of hydrogen bonds (neutral H-bond or salt bridge).

The final step in each thermodynamic cycle consists of the transfer of the His-Asp diadfrom water into the protein

FDPBcalculations

zwitterionic state (arbitrarily defined as the reference state in all calculations)

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pH dependence of the relative free energies of different protonation

states of the His235-Asp207 pair in protein environment

The titration curves for the His-Asp diads in HbHNL show the anionic state

in free enzyme has the lowest free energy except at very low pH

Very similar free energy of neutral and zwitterionic states equality of the pKas of

the two residues in diad, prerequisite for the formation of a short, strong hydrogen bond

(LBHB) and consistent with NMR dataFull agreement with NMR (one signal at 15.41 ppm between pH 4 and 9) and high-resolution

crystallographic evidence (density for one proton located near Nδ of His235)

pKa of His235 ~ 2.5

Free enzyme Bound enzyme

pKa of His235 ~ 10

NMR signal at 19.35 ppm diminishes above pH 7 with reappearance of 15.41 ppm signal.

No further change at pH > 9

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Protonation states for His – Asp diad

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Free enzyme; His235 pKa 2.5

Bound enzyme; His235 pKa 10





Implications

• His235 acts as the general base in HbHNL, because it catalyzes (via Ser80) the crucial step of cyanohydrin deprotonation.

– deprotonation must involve a substrate-induced pKa-shift of its side-chain,

– in the substrate-free form of the enzyme, the His235 side-chain is in partial contact with solvent and,

– if its pKa were already in the substrate-free form high enough to deprotonate the cyanohydrin OH (with a pKa ~ 10.7), His235 would become protonated by water.

• Substrate-induced pKa-shift is indicated by the free energy computations and the NMR results

• Inhibitor-induced equalization of the energy levels of zwitterionic and neutral protonation states is consistent with observation of a SSHB

• Free energy of the anionic state is increased by about 10.6 kcal/mol (i.e. the apparent pKa of His235 shifts from 2.5 to 10) in the substrate induced state.

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Summary• NMR experiments that show that the “catalytic” hydrogen bond converts

into a short, strong hydrogen bond upon addition of a strong competitive inhibitor mimicking the rate-limiting transition state of the enzyme reaction

• Poisson-Boltzmann computations based on NMR data show occurrence of the SSHB (LBHB) is paralleled by a catalytically relevant increase in basicity of the general base upon approach of the transition state

– finite difference Poisson-Boltzmann calculations yielded relative free energies of four protonation states of the His235-Asp207 pair in solution as well as in the protein environment with and without bound inhibitor – program DelPhi

– computations indicated a shift in the apparent pKa of His235 from 2.5 to 10

• increase in basicity is a prerequisite for His235 to act as general base in the HbHNL-catalyzed cyanohydrin reaction – note: pKa matching with Ser-O- alkoxide (pKa ≥ 15)

• Low-barrier hydrogen bonds are useful diagnostic tools to indicate the matching pKas of a hydrogen bonded donor-acceptor pair to rationalize enzymic mechanisms

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electrostatic perturbation in acetoacetate decarboxylase

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Significance• Acetoacetate decarboxylase (AADase) is a 365 kDa homododecameric enzyme that

catalyses the conversion of acetoacetate to acetone, is a key component in the anaerobic metabolism of carbohydrate in solventogenic bacteria.

– The enzyme was very much in demand in World War I because it was involved in a commercial process to make acetone, which was needed for airplane wing glue

• In the early 1960s, Westheimer used AADase to pioneer the application of methods in physical organic chemistry to the study of the chemical and catalytic mechanism of enzymes.

– These studies revealed that the mechanism of AADase proceeds through a Schiff-base intermediate formed by reaction of Lys 115 with substrate.

• A reporter group used to measure directly the pKa of Lys 115 revealed it to be 5.95, a value 4.5 orders of magnitude below that expected.

– Westheimer hypothesized that the pKa of Lys 115 was electrostatically perturbed by charge–charge repulsion due to the proximity of the protonated ϵ-amino group of an adjacent Lys 116

– This marked the first appearance of the proposal of microenvironment effects in enzymology

• The electrostatic perturbation hypothesis has been demonstrated in a number of enzymes, but never for the enzyme that inspired its conception, owing to the lack of a three-dimensional structure.

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Catalytic mechanism

• Acetoacetate decarboxylasecatalyses the decarboxylation of β-keto acids

• first step of the reaction is the formation of a Schiff base

• Schiff base formation creates an electron sink allowing rapid decarboxylation

• Nucleophilic attack occurs via Lys-115 on the substrate ketone to form a Schiff base

• Maximal activity at pH 5.95

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Ho MC, Ménétret JF, Tsuruta H, Allen KN. The origin of the electrostatic perturbation in acetoacetate decarboxylase. Nature. 2009 May 21;459(7245):393-7. substrate analogue

2,4-pentanedione





X-Ray crystal structure of AADase liganded to 2,4-pentanedione

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Lys-115 nucleophile at active site bottom Schiff base formed with substrate analogue: 2,4-pentanedione

Entrance to active site





Surface representation with 2,4-pentanedione bound in AADase active site tunnel

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Positions of Lys 115 and Lys 116 in active site

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The originally proposed pKa perturbation of Lys 115 by means of Coulombicdestabilization by a like-charged residue is not supported by this structure, because the ϵ-amino groups of Lys 115 and Lys 116 are separated by 14.8 Å

Highbarger LA, Gerlt JA, Kenyon GL. Mechanism of the reaction catalyzed by acetoacetatedecarboxylase. Importance of lysine 116 in determining the pKa of active-site lysine 115. Biochemistry. 1996 Jan 9;35(1):41-6.

site-directed mutagenesis and chemical rescue expts



Lys 115 and surrounding environment

• Hydrophobic environment of the active site, comprising Phe 26, Leu 71, Tyr 74, Met 96, Leu 98, Tyr 113 and Leu 233 destabilizes the protonated amine of Lys-115.

– Furthermore, the side chain of Lys 115 does not form any hydrogen-bonding interactions.

• Role for Lys 116 in the precise positioning of the nucleophilic Lys 115.

– consistent with previous studies including site-directed mutagenesis and chemical rescue that established the essentiality of the ϵ-amino group of Lys 116 in catalysis and the maintenance of the depressed pKa of the nucleophilic Lys-115

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Schiff base formation with 2,4 pentanedione

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Glu 76 side chain has alternative conformations: 1) 4.0 Å from the β-carbonyl group of the inhibitor, 2) 3.3 Å from the Glu 61 carboxylate positioned closer to the solvent channel opening



Proposed mechanism of AADase

• In the decarboxylation of β-keto acids, the bond undergoing cleavage must be out of the plane of the imine π bond.

• In the AADase enamine complex, the observed electron density for the 2,4-pentanedione shows that the proximity of Glu 76 would force the carboxylate group of substrate out of the plane of the enolic oxygen and Schiff-base nitrogen, favouring the expected geometry for decarboxylation.

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Summary

• The large shift of the pKa of Lys 115 of 4.5 pKa units is not due to the proximity of Lys 116 as had been thought

– Lys-116 side chain is oriented away from the active site

• Instead, Lys 116 participates in the structural anchoring of Lys 115 in a long, hydrophobic funnel provided by the fold of the enzyme

• Thus, AADase perturbs the pKa of the nucleophile by means of a desolvation effect by placement of the side chain into the protein core

– proximity of polar residues then facilitate decarboxylationthrough electrostatic and steric effects.

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