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Exploring Molecular Oxygen Pathways in Hansenula polymorpha Copper-containing Amine Oxidase Cuá

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Abstract The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels.
The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed.
Copper click at this page oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively.
The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches.
A crystal structure of H.
Calculated O 2 free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O 2 migration routes of differing probabilities within the protein matrix.
Copper amine oxidases are ubiquitous copper containing enzymes that oxidize primary amines to aldehydes through the reduction of molecular oxygen to hydrogen peroxide.
CAO catalysis is dependent upon the protein-derived cofactor 2,4,5-trihydroxyphenylalaninequinone TPQ.
The TPQ is derived from an endogenous tyrosine through a self-catalytic process requiring only molecular oxygen and Cu II see.
Hansenula polymorpha amine oxidase is the eukaryotic CAO that has been kinetically characterized in the most detail —.
HPAO follows a Bi Bi ping-pong reaction mechanism that can be expressed as two half-reactions, reductive and oxidative see.
In the reductive half-reaction the enzyme oxidizes a primary amine to an aldehyde, generating the 2e - reduced aminoquinol form of the cofactor.
In the subsequent oxidative half-reaction molecular oxygen is reduced to hydrogen peroxide via cofactor reoxidation to TPQ.
Biochemical studies from several different laboratories have led to mechanistic proposals for the catalytic cycle of CAOs .
These studies have given significant insight into the mechanism for the reductive half-reaction.
However, the details surrounding the activation of molecular oxygen both in terms of the biogenesis of the TPQand of the catalytic oxidative half-reaction, remain the subject of intense study —.
The utilization of copper as a redox center has been the focus of recent controversy.
Because CAOs contain a copper ion in their active site, chemical intuition suggests Cu I as the O 2-activating species to give Cu II -superoxide.
Upon anaerobic amine reduction of CAOs, just click for source equilibrium between Cu II -aminoquinol and Cu I -semiquinone is observed, with Cu I -semiquinone yields varying from 0 to 40% depending on the enzyme source .
Plant CAOs have high yields of Cu I -semiquinone and bacterial CAOs are in the middle of the range, whereas non-plant eukaryotic CAOs have minimal or undetectable amounts of Cu I after amine reduction.
It has been postulated that despite the unobservable amount of Cu I -semiquinone in many non-plant eukaryotic CAOs, there must still be enough Cu I present for this to be the O 2-activating species during catalysis.
However, Co II -substituted HPAO has a k cat almost identical to Cu-HPAO at pH 7 .
Additionally, kinetic isotope effects, steady state kinetics, viscosogen, and stopped-flow experiments have shown that the reduction of molecular oxygen contributes 29% to the overall k cat .
This is a surprising result because the reduction of O 2 by Cu I to give superoxide is likely to be extremely fast.
Based on these results a new mechanism was proposed for both HPAO and bovine serum amine oxidase that invoked an off-metal molecular oxygen binding pocket for the first electron transfer to O 2 directly from the aminoquinol.
The off-metal binding site at which O 2 undergoes the initial one-electron reduction was proposed to be adjacent to the metal, bounded by the side chains of Met-634, Leu-425, and Tyr-407.
Xenon complexation can be used to probe the interior of protein structures for sites that favor molecular oxygen gas binding.
Because of its analogous properties in size and hydrophobicity, any region that binds xenon is proposed to also be favorable for O 2.
In myoglobin, for instance, xenon binding cavities observed crystallographically were also shown to bind photolysed CO, another dioxygen mimic —.
X-ray crystal structures of CAOs bound to Xe are available from bacterial Arthrobacter globiformis, AGAOyeast Pichia pastoris, PPLOplant Pisum sativum, PSAOand mammalian sources bovine serum amine oxidase.
An additional solution phase NMR study found that Xe also bound to lentil seedling CAO Lens esculentaand the data implicated a Xe-induced conformational change at the active site during amine reduction.
The anteroom is too far from either the copper or aminoquinol to be directly involved in the reduction of molecular oxygen to superoxide but could act as a holding pocket for O 2 close to the active site, and is consistent with results that indicate O 2 is pre-bound to the enzyme before reduction .
The off-metal hydrophobic pocket proposed through mutagenesis studies at Met-634 in HPAO is adjacent to the anteroom, and the two pockets share a common residue, Leu-425 see.
The inland lake is conserved at the dimer interface of CAOs and has been suggested as the entry point for O 2.
However, as a polar channel often only one water molecule wide, it is hard to reconcile this with the hydrophobic nature of molecular oxygen.
To help resolve all these data, we wanted to delineate more clearly molecular oxygen holding pockets in HPAO as well as define other potential molecular oxygen pathways into the buried active site.
Here we report a crystal structure of HPAO in complex with xenon that offers new insights into CAO-molecular oxygen interactions.
Bound xenon in the dense hydrophobic interior of the catalytic domain suggests a putative channel within CAOs accessible to molecular oxygen.
Free energy maps of molecular oxygen, computed from dynamics simulations of several CAOs in the absence of xenon, correlate with experimental observations and suggest new pathways that molecular oxygen might take to the buried active site.
EXPERIMENTAL PROCEDURES HPAO Purification—Wild-type HPAO wtHPAO for crystallization was heterologously expressed in Saccharomyces cerevisiae and purified as described previouslywith modifications.
The preparation of the URA - media contained 1.
Previous protocols called for 6.
The soluble fraction of the freshly lysed cells was not dialyzed as previously reported but was, instead, equilibrated into 5 m m potassium phosphate buffer by 10-fold dilution.
The diluted soluble fraction was then immediately loaded onto the DEAE anion exchange column.
This was followed by size exclusion chromatography on a Sephadex S-300 column.
The protein was buffer exchanged into 50 m m HEPES, pH 7.
The ratio of protein to well solution in the hanging drops was 1:1, with volumes of 2.
The best diffracting crystals were cube-shaped with dimensions of 40 × 40 × 30 μm and grew within 3 days.
The space group of the crystals used in this study was P2 1, although crystals also grew in the space groups C2 and P2 12 12 1 from the same crystallization conditions.
Cryoprotection was performed by soaking crystals for 10 s in 25% high purity glycerol Hampton Research mixed with mother liquor directly from the crystallization well.
Xenon Complexation—Crystals of HPAO in cryoprotected mother liquor were complexed with xenon using a pressure cell Rigaku at a pressure of 10 atm for 10 min.
To prevent dehydration of the crystal during the pressurization, a small knot of water-saturated Kimwipe was placed at the bottom of the chamber near where the loop containing the crystal would rest during the pressurization.
Before pressurization the crystals were soaked in cryoprotectant.
Within 5—10 s of depressurization the crystals were flash-frozen in liquid nitrogen.
Spectra were generated from the integration of 10 19-ms exposures.
Structure Determination and Refinement—Each x-ray data set was collected from a single crystal at 100 K at the Advanced Photon Source, Argonne National Laboratory Beamline ID-19, SBC-CAT.
For the xenon complex, data were collected at two detector distances; one for the optimal collection of high resolution data and another for the minimization of overlaps at lower resolution.
These two data sets were merged for optimal completeness.
Data were processed with HKL2000 and SCALEPACK.
Molecular replacement was performed with MOLREP, part of the CCP4 suiteusing the previously deposited HPAO model, Protein Data Bank PDB code 1a2v.
Calculation of the anomalous map was performed using the programs of the CCP4 suite.
Model building was performed using COOT.
Refinement of the model was performed using REFMAC5.
There was no use of non-crystallographic symmetry restraints during refinement.
Atomic coordinates for the position of xenon atoms were located by 6-fold averaging of the anomalous map.
Individual Xe occupancies were set at the occupancy that equated Xe B-factors to those of the surrounding protein during refinement and absence of F o - F c electron density.
Mutagenesis—Mutations were made to the pDB20-HPAO plasmid using the Stratagene QuikChange kit.
Primers were purchased high performance liquid chromatography-purified from Operon.
The forward primers are given below; the reverse primers were complementary to these.
The mutated codon is given in bold, and the changed bases are underlined.
Sequences were confirmed by automated DNA sequencing University or California, Berkeley, CA.
The mutated plasmids were transformed into the S.
Mutant Characterization—TPQ content was determined by titration against phenylhydrazine in 100 m m KP i, pH 7.
TPQ per subunit concentrations were then calculated using a total protein concentration obtained from a Bio-Rad protein assay using a bovine serum albumin standard and a M r of 75,700 for HPAO.
Metal content was determined by inductively coupled plasma-atomic emission spectroscopy ICP-AES using a PerkinElmer Life Sciences Optima 3000DV spectrometer, analyzing the copper wavelengths at 327.
Electron Paramagnetic Resonance—Electron paramagnetic resonance EPR spectra were collected at 15 K using a Varian E9 spectrometer with a scan range of 1790—4790 G, microwave power 5 milliwatt, microwave 9.
A buffer blank spectrum was subtracted from sample spectra before further processing.
Steady State Kinetics—Steady state kinetic measurements were carried out by monitoring oxygen consumption using a Clark electrode and a YSI-5300 biological oxygen monitor.
Standard conditions were: final volume, 1 ml; temperature, 25 °C; reactions initiated by the addition of HPAO.
Solutions were equilibrated to atmospheric conditions by stirring at 1000 rpm for 5 min just before initiation.
For reactions at different oxygen concentrations, two flow meters were used to regulate the flow of O 2 and N 2, and the equilibration time was extended to 10 min.
For assays in the pH range 6—8 and 8—9, 100 m m KP i and 25 m m pyrophosphate buffers were used, respectively.
The ionic strength of all buffers was kept constant at 0.
Data were fit directly to the Michaelis-Menten equation, and k cat was calculated using the active protein concentration as determined by phenylhydrazine titration.
O 2 Free Energy Maps—Computational analysis was used to predict the location of preferred pathways taken by O 2 to reach the active sites of HPAO, AGAO, PPLO, and PSAO.
The starting co-ordinates for each simulation were the native oxidized structures and, thus, contained no information regarding experimentally determined Xe binding sites.
The protein PDB coordinates Apologise, luxembourgで利用可能なfantasmaによるゲームを特徴とするカジノ this, 2oov; AGAO, click the following article PPLO, 1w7c; PSAO, 1ksi were first solvated in a 50 m click NaCl water box.
Keeping everything else fixed, the solution was then equilibrated for 30 ps followed by a combination of the solution and protein side chains for 50 ps and, finally, the entire system for 950 ps using the NAMD simulation program.
Trajectories were then recorded for 10-ns simulations performed at constant pressure 1 atm, Langevin piston and temperature 300 K, Langevin dynamics using long-range PME electrostatics and the CHARMM22 force-field with custom parameters built by analogy for the TPQ residue and histidine-copper bonds.
The trajectories were used as input for an implicit ligand sampling analysis contained in the VMD software packageresulting in detailed three-dimensional free energy profiles for O 2 placement inside the protein based on the assumption that the presence of gas molecules can be treated as a weak perturbation to protein natural thermal motion.
RESULTS Diffraction data were recorded to 1.
Both unaveraged and 6-fold-averaged anomalous electron density maps revealed four major xenon binding sites per HPAO monomer see.
Xe1 is the strongest anomalous peak and lies within the center of the large β-sandwich of the catalytic domain see.
Xe2 and Xe3 see lie in the amine substrate entry channel that leads from the surface of the molecule to the reactive O5 position of the TPQ.
The weakest anomalous peak, Xe4, was detected near Pro-225 at the surface of HPAO.
Measurement of the absorbance spectrum from the crystal after x-ray data collection revealed a characteristic 480 nm absorbance peak ensuring that it remained oxidized throughout exposure to the x-irradiation supplemental Fig.
We have also solved the oxidized structure alone to 1.
Notable structural features include an active copper-off TPQ orientation in one of the three dimers present in the asymmetric unit, whereas the other two dimers have mixed TPQ orientations composed of both active copper-off and inactive copper-on TPQ axially ligated to copper via O4 conformations and were modeled as such.
The copper-off active sites also show the presence of an oxidized methionine Met-634 near the Cu II ion, with no evidence of oxidation at any other methionines in the structure.
This site is probably particularly sensitive to radiation damage due to its proximity to the redox active site of the enzyme.
It is difficult to judge whether Met-634 is oxidized in the subunits with both copper-on and copper-off TPQ conformations because the copper-on form obscures the electron density of that position.
Reactions catalyzed by copper amine oxidases are biogenesis a and catalysis b.
P represents the rest of the polypeptide chain.
R represents the moiety of the substrate amine, which varies from a hydrogen atom to a polypeptide.
Modeled regions of xenon binding in HPAO.
The orange sphere represents a putative site suggested in HPAO by mutational and kinetic analysis.
The cyan sphere represents the consensus Xe binding site found in AGAO, PSAO, PPLO, and bovine serum amine oxidase surrounded by the anteroom residues, colored in green .
Waters shown in small red spheres and connected by dashed black lines indicate the path from the active site to the inland lake.
The figure was generated using Pymol.
To further investigate the relevance of xenon binding pockets in CAOs in influencing reactivity with molecular oxygen, we designed several point mutations at residues around these sites.
The residue Leu-425, which divides the two proposed O 2 binding pocketswas mutated to phenylalanine to complement the previously check this out L425A mutant and investigate the effects of increasing residue size and hydrophobicity at this position.
Ile-622 is a possible candidate residue for controlling entry of gaseous molecules into the anteroom from the inland lake, and so this was mutated to a tyrosine in an effort to block entry to this site.
The side chain of Ile-639 sits in the anteroom and is comparatively remote from the copper, so this residue was mutated to a phenylalanine in an attempt to restrict gas binding.
Finally a L643F mutation was made at the Xe1 site in the hydrophobic core of the catalytic domain in an attempt to disrupt gas conduction through this region.
TABLE 2 Steady state kinetic measurements on wild-type HPAO and mutants All measurements carried out at pH 8 and 25 °C.
The Cu II EPR spectra of L425F, I639F, and I622Y were all similar to wtHPAO, indicating that the primary coordination environment of the copper had not been perturbed supplemental Table S3.
The limiting rate constant, k cat, for all the mutants was reasonably similar to that of wt, with the slowest being down not more than 2-fold.
This is to be expected as these mutations are remote from the site where the chemistry for this step occurs.
The p K a values of 6.
For both L425F and I639F, the data fit well to a 2 p K a model.
For I639F the p K a values 6.
The altered p K a values for L425F indicate that this mutation is directly influencing the active site while that of I639F is not.
The migration of molecular oxygen through CAOs from a protein-wide perspective was explored using a computational method called implicit ligand samplingdeveloped for finding gas migration pathways inside proteins.
This method takes advantage of the fact that O 2 travels along transient cavities in the protein that continuously form and disappear over relatively short timescales 1 ns.
By monitoring these transient cavities as they appear over a 10-ns molecular dynamics window in the protein, even in the absence of O 2, the potential of mean force PMF corresponding to the placement of O 2 everywhere inside the protein can be computed.
The result is a complete three-dimensional map of the free energy of placing an O 2 molecule anywhere inside a protein.
Networks of potential O 2 entry and exit pathways inside the protein are inferred by connecting favorable areas for O 2 that are in close proximity to one another forming gas migration pathways.
Because the thermal fluctuations responsible for the migration of gas molecules such as O 2 inside proteins are well sampled during the simulation time used 10 nsthe computed O 2 pathways are highly probable with the caveat that possible O 2 pathways which are caused by rare events may not be identified by this method.
This approach has been applied here to HPAO, AGAO, Cuá, and PSAO to correlate the experimentally observed xenon sites with calculated pathways and supplemental data files pmf-xxxx-o2—10ns.
Although the starting models in each case had TPQ modeled in the active copper-off conformation, where TPQ hydrogen bonds via its O 2 position to the axial water ligand of agree, 【emoji planet】絵文字をテーマにした連鎖系の愛されスロット。ゴンゾーも登場!? think copperin AGAO and PPLO the TPQ migrated onto the copper during the 1-ns equilibration that is part of the free energy calculation.
This copper-on form, in which the TPQ displaces the axial water ligand to directly ligate to the copper via its O4 position, is a common conformation observed in CAO crystal structures including the HPAO structures presented here and is required for the biogenesis of TPQ .
None of the calculated pathways were affected by this change in TPQ conformation.
The location of experimental Xe binding correlates well with the locations 【ポケモン赤・緑・青・ピカチュウ】パラセクトの種族値と入手方法 minimum free energy computed from implicit ligand sampling on a 10-ns molecular dynamics simulation in each CAO supplemental Fig.
The only exceptions are surface-bound Xe sites Xe4 in HPAO and Xe903 in AGAO.
In addition, implicit ligand sampling provides a complete three-dimensional map for the migration of O 2 between favorable regions.
This map is created by contouring energy isosurfaces representing elevated PMF values 1.
In HPAO, we identify two major regions that contain the most probable pathways from protein solvent boundary to the buried active site according to the implicit ligand sampling calculation.
One starts from the amine entry channel close to the 5 position of the TPQ cofactor where the reductive half-reaction chemistry is known to take place left pathway.
Another leads through the hydrophobic core of the β-sandwich toward the active site right pathway.
The two pathways merge near the active site, and there is an energetically favorable O 2 docking site that could play a role in pre-binding O 2 in HPAO before it enters the active site for activation.
The PMF maps of AGAO, PPLO, and PSAO have a larger less favorable PMF value in the area of the HPAO docking site, and in these enzymes the anteroom is the most favorable area close to the active site for pre-binding O 2.
The AGAO PMF maps suggest that the most favorable migration route is from the D3 β-sandwich hydrophobic core through the anteroom to the active site, which is similar to that suggested by the HPAO PMF maps.
In PPLO and PSAO, passage via this route appears much less favorable.
In all cases the short polar channel that exists between the inland lake and the active site, which had previously been proposed as the probable entry site for molecular oxygen, is not the only pathway between these two areas.
Rather, there are connections from the inland lake to the anteroom, particularly in PPLO and PSAO where an equally favorable route appears to occur near, but distinct from the previously described polar channel.
Xenon binding sites in HPAO.
One monomer is displayed and colored by domain: blue, D1; yellow, D2; green, D3.
The monomer displayed in Cα trace has the TPQ in stick, and the copper as a green sphere.
Xenons are displayed as magenta spheres, and the numbering corresponds to peak size in the anomalous map.
The black mesh represents the anomalous map contoured at 9.
Residues with side chains within van der Waals contact are displayed.
Residues in gray belong to the HPAO + Xe complex, and those in blue belong to oxidized HPAO.
The figure was generated using Pymol.
DISCUSSION The location of Xe1 in the HPAO + Xe complex has revealed the presence of a binding region that is not accessed by a traditionally defined channel.
Xe1 represents ligand binding in a solvent-inaccessible void.
The puzzling nature of this singular site in HPAO provoked us to look for similar sites in other CAOs.
In cuá a composite overlay of all available xenon complex data, totaling cuá different species, we were immediately struck by the chain of Xe atoms that occupied the hydrophobic core of the catalytic β-sandwich.
The overlaid sites are not coincident, yet they appear to mark a highly favorable area for a small hydrophobic molecule, such as molecular oxygen, to reside.
This chain of Xe atoms also appears to indicate a pathway whereby molecular oxygen might enter at either end of the catalytic β-sandwich and then proceed toward the active site, click through or close by the anteroom along the way arrows.
Indeed, the lining of the catalytic β-sandwich with larger hydrophobic residues well suits it for the favorable accommodation and conduction of molecular oxygen.
Large hydrophobic side chains, like those found in this region, are highly flexible and mobile, offering a higher propensity for packing defects that allows the passage of small hydrophobic molecules.
The sequence conservation in this region for the CAOs is similar to that seen for any large protein hydrophobic core, suggesting there is nothing special about the CAO catalytic β-sandwich in terms of O 2 migration.
Implicit ligand sampling performed in CAOs with a molecular oxygen probe.
Shown are PMF maps of HPAO bAGAO dPPLO eand PSAO f.
Blue transparent represents a 1.
Black mesh represents a 3.
Most favorable routes for O 2 from the surface are marked by green lines.
Residues shown include the TPQ and histidine ligands of the Cu II shown as a green sphere.
The box in the HPAO map b is enlarged c to show a close-up of the HPAO active site with PMF map displayed.
Surrounding residues are colored in purple and green, corresponding to the energetically favorable HPAO O 2 binding site and the anteroom, respectively.
An additional striking feature of the CAO + Xe composite overlay was the presence of Xe atoms in the amine channel.
In the HPAO + Xe complex the amine channel supports the binding of two atoms of xenon in place of ordered waters normally found in the oxidized structure Fig.
AGAO provides an additional site nearer the surface of the enzyme.
As part of the amine channel, these observed sites are on the opposite side of the cofactor from the location of dioxygen chemistry.
In addition to marking a region suitable for O 2 binding, the closest peak to the TPQ in HPAO, Xe3, probably indicates a favorable binding site for the aliphatic portion of the small amine substrates favored by this CAO and helps orient the substrate for correct nucleophilic attack at the C5 of TPQ.
In HPAO, orientation of substrate would be mediated through a favorable hydrophobic interaction with Cuá />By structural overlay, the position of Trp-156 aligns it with gating residues found in other CAOs.
In the case of ECAO, Tyr-381 is known to stack on the aromatic portion of its preferred aromatic monoamine substrates and has a stabilizing and orienting effect.
Because no side-chain movement is involved in the binding of Xe2 in HPAO, Trp-156 does not seem to act as a gate per se, being already in the open conformation.
Comparison of CAO xenon binding sites.
The backbone of HPAO is displayed and colored by domain: blue stick, D1; yellow stick, D2; green ribbon, D3.
Xenon sites from individual complexes are coded by color red, PSAO PDB code 1w2z ; yellow, PPLO PDB code 1rky ; blue, AGAO PDB code 1rjo magenta, HPAO PDB code 2oqeexcept surface-bound xenon sites, which are displayed as gray spheres.
The top inset shows the amine channel.
The bottom inset shows a proposed molecular oxygen pathway identified in this study.
The active site is shown in stick, and the copper is shown as a green sphere.
Only the xenon sites in the article source D3 β-sandwich and close to the anteroom are shown.
Colors are the same as in a, and arrows indicate the direction of molecular oxygen movement to the active site using the proposed pathway.
The figure was generated using Pymol.
The analysis of putative molecular oxygen pathways was taken one step further by the generation of PMF maps through implicit ligand sampling analysis.
PMF maps reveal two important features present in protein structures.
The first feature is the location of hydrophobic binding sites in CAOs that are favorable for small hydrophobic molecules and which may or may not correspond to preexisting static cavities.
The second feature is the lowest energy route available between determined binding sites by generating isosurfaces at elevated PMF levels.
The free energy map calculation in HPAO confirms with excellent fidelity the experimentally observed xenon binding sites across CAOs within the core of the catalytic β-sandwich supplemental Fig.
By elucidating several regions within the catalytic β-sandwich D3 domain that are favorable and accessible to molecular oxygen binding, we can visualize a series of binding sites potentially used en route to the active site.
Similar binding possibilities in the β-sandwich are seen in map calculations for AGAO, PPLO, and PSAO, with a route being most apparent in AGAO.
The molecular oxygen pathways vary slightly between CAOs, especially as they near the active site, and suggest that use of the anteroom is not an absolute requirement.
However in PPLO, PSAO, and AGAO, where utilization of the anteroom has been suggested by Xe complexation, the free energy maps suggest a high probability for O 2 binding in that region.
The HPAO PMF maps revealed an equally energetically favored O 2 pathway to the buried active site through the amine channel.
In the case of a partially polar molecule, such as the amine, this pathway terminates near the O5 position of the cofactor.
However, when considering O 2, PMF maps show that the path instead continues past the backside of the TPQ and merges with the β-sandwich route near an HPAO-specific binding site.
Due probably to the HPAO narrow amine channel structure and the presence of some hydrophobic residues, this channel is equally suited 【fgo】カーマの性能詳細と評価 conduct O 2 as well as amine substrate.
Modeling of methylamine and ethylamine Schiff base intermediates from the reductive half-reaction did not interfere with the proposed O 2 pathway from the amine channel, although the Schiff base with benzylamine did .
However, because benzylamine is a poor substrate for the enzyme, with a 100-fold decrease in k cat compared with ethylamine, it is not thought to be a physiologically relevant substrate.
So for the preferred small aliphatic substrates of HPAO, there appears to be no step of the catalytic cycle when the amine channel would not be available for O 2 migration.
Originally, the entry point for molecular oxygen to the active site of CAOs was proposed to be from the inland lake.
Because the PMF maps confirm this region as a prime reservoir for molecular oxygen, it seems likely it could act as a source of O 2.
The previously proposed narrow polar channel that has been described between the active site and the inland lake does appear to have some probability of acting as an entry point for O 2 in AGAO, PSAO, and PPLO based on the implicit ligand sampling analysis, although other connections from the inland lake are also evident.
In the case of HPAO, however, this path is energetically unfavorable for O 2purple.
As noted by Duff et al.
This route seems at odds with one meant to deliver O 2 to a site of pre-binding before activation at the active site.
These factors cause us to favor describing the previous proposal primarily as an exit channel for the product H 2O 2, benefiting both from its polar nature and short length, which reduces the possibility for oxidative damage to the enzyme during exit of this reactive molecule.
Taken together, the I639F and I622Y mutations, the PMF maps, and the lack of xenon in this region imply that the anteroom is not a significant region for O 2 pre-binding in HPAO.
This is different from AGAO, PSAO, and PPLO, where xenon complex data and implicit ligand sampling support a role for the anteroom.
The L643F mutant of this study, which was designed to alter the side-chain size and flexibility within the hydrophobic core of the catalytic β-sandwich at the HPAO Xe1 site, also had no effect on O 2 kinetics.
This is consistent with the probability that there are multiple paths to the active site, including in HPAO via the substrate amine channel.
Further studies are required to assess how important the favorable area within the D3 β-sandwich is for O 2 channeling in HPAO, but our results indicate that single or even double point mutants may do little to significantly affect oxygen kinetics.
Kinetic data support O 2 being pre-bound to the enzyme before activation, which could involve one or more favorable holding areas for molecular oxygen distinct from the site of O 2 activation.
We propose that the catalytic β-sandwich and amine channel, in addition to the inland lake, serve as reservoirs of molecular oxygen, containing multiple pre-binding sites en route to the active site.
In this way, multiple regions of the protein act as internal reservoirs for small hydrophobic gases, continuously supplying the active site with molecular oxygen.
The apparent flexibility in approach routes for molecular oxygen in the different CAOs suggests that initial entry may not be at a single location.
Instead, it is better viewed as pathways of differing probabilities where O 2 is transiently held before movement to the pocket containing Met-634, which still remains the more info candidate site for off-copper activation.
The accumulated evidence from crystallography, computational, and biochemical studies on several different protein systems supports the notion that there are specific pathways for gas migration through the protein matrix—.
The combination of xenon complexation and implicit ligand sampling calculations on CAOs agrees with this premise, cuá that molecular oxygen migration appears to follow preferred routes.
There is some conservation in the pathways for molecular oxygen migration in CAOs, although their importance varies from one species to another.
In particular, the hydrophobic core of the D3 catalytic β-sandwich can act as a reservoir for molecular oxygen, with O 2 then moving into the vicinity of the active site for eventual activation.
The presence of routes is largely dictated by the overall architecture of the protein, where certain secondary and tertiary structural folds, such as the hydrophobic core of the β-sandwich found in CAOs, can create favorable reservoirs for small hydrophobic molecules such as molecular oxygen.
Although the overall fold is conserved in the catalytic β-sandwich, differences in the primary amino acid sequence probably account for the species-specific differences in opinion, 白鳥詩織(cv:高橋美佳子) 生徒名簿 consider pathways.
Implicit ligand sampling analysis, by monitoring transient level of cavity formation, allows the accurate prediction of hydrophobic binding regions that match with high fidelity the experimentally observed xenon binding sites.
Because there appear to be multiple routes in any given CAO, it is unlikely that a single site-directed mutant is going to impact molecular oxygen access, and this fits with the data in this study.
Molecular oxygen pathways, unlike many other substrate channels, seem to rely on a delicate balance of tertiary structure, side-chain hydrophobicity, and conformational flexibility to allow the efficient transport of this small hydrophobic molecule.
Acknowledgments We thank Teresa De la Mora-Rey for help during x-ray data collection as well as the staff of Structural Biology Consortium-Collaborative Access Team, especially Steve Ginell.
We also thank Patton Fast of the University of Minnesota Supercomputing Institute for computer support.
https://casinobonusgamesonline.com/37/2384.html of the Advanced Photon Source was supported by the United States Dept.
In-house x-ray data collection was supported by a Minnesota Partnership for Biotechnology and Medical Genomics Grant SPAP-05-0013-P-FY06.
Computer resources were provided by the Basic Sciences Computing Laboratory of the University of Minnesota Supercomputing Institute.
The costs of publication of this article were defrayed in part by the payment of page charges.
Section 1734 solely to indicate this fact.
S1—S3, and data files.
Crystallogr 50, 760-763 Murshudov, G.
B 102, 3586-3616 Kim, M.
M701308200 June 15, 2007 The Journal of Biological Chemistry 282, 17767-17776.

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