my work at ohsu
DESCRIPTION
A presentation of my work on peptidyl-hydroxylating monooxygenase conducted at Oregon Health and Science University.TRANSCRIPT
- 1. Production and Mechanistic Characterization of Peptidylglycine Hydroxylating Monooxygenase (PHM) Andrew Bauman Senior Research Associate @ OHSU
- 2. Function of PHM and its partner PAL Vederas, J. C. et.al . J. Chem. Soc., Chem. Commun. , (1991) 571-572. Eipper, B. A. et. al ., Biochemistry, 41 (2002) 12384-12394.
- 3. Structure of PHMcc (aa 42 356)
- 4. PHM, A Copper Monooxygenase Cu H H172 H108 H107 H244 H242 Di-I-YG Substrate Cu M Y318 R240 N316 D1 D2 Q170 Amzel, L. M. et. al ., Science, 278 (1997) 1300-1305. Substrate C is in close-proximity to Cu M Cu M is the site of dioxygen binding and catalysis. S = C-terminal D-aminoacid
- 5. Active Oxidized State of PHM
- 6. General PHM Mechanism
- 7. Active Site Coordination of PHM at Different Stages (b)
Reduced State
- M314 is not coordinated in
- the oxidized state
- 8. Proposed Mechanisms and Intermediates
- Substrate mediated pathway
- Superoxide channeling
- Peroxide intermediate
- Superoxide intermediate
- 9. Substrate-Mediated Electron Transfer Amzel, L. M. et. al ., Science, 278 (1997) 1300-1305.
- 10. Superoxide Channeling Mechanism Proposed by Blackburn &
et al.
- Superoxide forms at the Cu H site Channels to the CuM site
- Cu M site supplies a proton and an electron to the superoxide converting it to hydroperoxide
- Hydroperoxide hydroxylates the substrate
- 11.
- Methods for obtaining a reliable supply of PHM and its mutants
- The spectroscopic and electronic description of intermediates
- The strong preference for methionine coordination at the oxygen
- activating Cu M center
- The pathway of electron transfer (ET) from the H to M site
- 12. Bauman, Andrew, T.; Blackburn, Ninian, J.; Ralle, Martina. Large Scale Production of the Copper Enzyme Peptidylglycine Monooxygenase Using an Automated Bioreactor. Protein Expr. Purif. (2007), 51(1), 34-8. Bauman, Andrew, T.; Jaron, Shula; Yukl, Eric, T.; Burchfiel, Joel, R.; Blackburn, Ninian, J. pH Dependence of Peptidylglycine Monooxygenase. Mechanistic Implications of Cu-Methionine Binding Dynamics. Biochemistry. (2006), 45(37), 11140-50. Bauman, Andrew, T.; Yukl, Erik, T.; Alkevich, Katsiaryna; McCormack, Ashley; Blackburn, Ninian, J. The Hydrogen Peroxide Reactivity of Peptidylglycine Monooxygenase Supports a Cu(II)-Superoxo Catalytic Intermediate. J. Biol. Chem. (2006), 281(7), 4190-8. Bauman, Andrew, T.; Boers, Brenda.; Blackburn, Ninian, J.; Characterization of the Peptidylglycine Monooxygenase M314H Mutant. New Insights Into Methionine Coordination, Oxygen Binding, and Electron Transfer. In preparation. Publications
- 13. Experiments Stopped-Flow Spectrokinetic Analyzer
- 14. Experiments Freeze Quench Spectrokinetic Analyzer
- 15. Experiments Dissolved Oxygen Electrode
- 16. Electron Paramagnetic Resonance Experiments
- 17. Experiments EXAFS Shell R( ) 2 2 ( -1 ) 2.5 N(im) 1.97 0.009 1.5 O/N 1.97 0.009 Cu N1 C2 C5 N4 C3 Cu N1 C2 C5 N4 C3
- 18. Large Scale Production of PHM
- PHM has not been successfully expressed in yeast or bacteria
- Proposed experiments required gram quantities of enzyme
- PHMcc successfully expressed in CHO cells
-
- CHO sells which secrete PHM grown in hollow fiber bioreactors
-
-
- Small manual bioreactor (B1)
-
-
-
- Large automated bioreactor (B2)
-
-
- Harvest media containing apo-PHM is collected and purified
- 19. Production of PHM Harvest media Ammonium Sulfate Gel Filtration Anion Exchange Reconstitution Experiments
- 20.
- Cells grow in the extra capillary space (ECS) of a capillary cartridge (Brx)
-
- Fed through the intercapillary space (ICS) by media pumped from
-
- a 1L reservoir
-
-
- 4 kDa cutoff allows passage of nutrients while retaining
-
-
-
- secreted PHM
-
- Housed in a sterile CO 2 incubator
-
- operated at ~ 5% CO 2 and 37 0 C
-
- Crude pH control using bicarbonate buffer and CO 2
- Required daily, manual Harvest
-
- compromised sterility
-
- increased residence time of PHM in the reactor
- 21. Problems
- S mall size of the Brx resulted in proportionally small yield
- Contamination and clogging led to short run lifetimes
- Enzyme Degradation
-
- Decreasing activity, Cu/Protein ratio, and solubility
-
- Clipping at Ser 61
-
-
- Increased exposure to high temperatures, proteases etc.
-
-
-
- pH fluctuations from 7.5 to 6.4 between feeding and harvest
-
- 22. B2 Schematic of B2 (Accusyst Minimax)
- 23.
- 24.
- 25.
- 26. Advantages
- Large size leads to higher production levels
- Continuous harvest into a refrigerated bottle
-
- less likely to compromise sterility
-
- lower residence time of PHM in bioreactor
-
- harvest media stored at 4 0 C
- Feedback control maintained optimal pH
- ECS loop pumps allows addition of serum and high MW nutrients
- 27. Quality Comparison of B1 and B2
- 28. Quality Comparison of B1 and B2
- 29. MALDI-MS of PHM from B2 ESI-MS of reduced/alkylated PHM provided evidence of an intact N-terminus 35,625 daltons was observed ~ (35,048 Da + (10*58 Da)) Quality Control of PHM from B2
- 30. Visible spectrum of PHM pH 8.0 Quality Control of PHM from B2
- 31. Description of Intermediates
- CuM(II)-peroxo is one potential intermediate
-
- 2 electron reduced species
-
- peroxide shunt should be possible
- 32. Substrate: Dansyl-Y-V-G Mix: Buffer pH 5.5, 5uM Cu++, 5uM PHM, Catalase PHM Reaction Mix Reductant TFA Quench every 30s Initiate Reaction RPHPLC Equipped with Fluorescence Detector Monitor Oxygen Consumption Quench entire reaction with TFA Peroxide Concentration Assay
- 33. Dissolved Oxygen Electrode Standard Hydrogen Peroxide Reaction + Oxygen Consumption
- 34. Standard Reaction Using Ascorbate as Reductant Substrate: Dansyl-Y-V-G Buffer pH 5.5, 5uM Cu++, 5uM PHM, 1mM ascorbate TFA Quench every 30s Add substrate to 300 uM RPHPLC equipped with Fluorescence Detector
- 35. Substrate: Dansyl-Y-V-G Standard Hydrogen Peroxide Reaction + HPLC Buffer pH 5.5, 5uM Cu++, 300uM substrate, 5uM PHM TFA Quench every 30s Add H 2 O 2 to 1mM RPHPLC equipped with Fluorescence Detector Peroxide Concentration Assay
- 36. Substrate: Dansyl-Y-V-G Standard Hydrogen Peroxide Reaction + Oxygen Consumption Quench entire reaction with TFA Buffer pH 5.5, 5uM Cu++, 300uM substrate, 5uM PHM Monitor Oxygen Consumption Add H 2 O 2 to 1mM Peroxide Concentration Assay
- 37.
- Catalysis occurred using peroxide as the only oxygen source
-
- H 2 18 O 2 experiments in the presence of
-
- 16 O 2 resulted in only 35% incorporation
-
- anaerobic conditions or under 18 O 2
-
- resulted in 90% incorporation ruling out
-
- solvent exchange
- 38.
-
-
- Two possible explanations for the data:
-
-
-
-
- 1. Generation of an enzyme intermediate capable of exchange with
-
-
-
-
-
- atmospheric dioxygen
-
-
-
-
-
- 2. Simple reduction of the Cu(II) centers by peroxide and subsequent
-
-
-
-
-
- reaction with solution dioxygen
-
-
-
-
-
- Strict anaerobic conditions are difficult to achieve
-
-
-
- 39. oxygen evolution from peroxide measured in the O2-electrode under different conditions. Initial trace , 100 mM MES pH 5.5, 5 M Cu2+ and 5 M PHM; A , addition of 1 mM H2O2; B , addition of 200 M dansyl-YVG substrate. Evolution of Oxygen From Peroxide and PHM
- 40.
- 41. Substrate: Dansyl-Y-V-G Peroxide Generation by Glucose/Glucose Oxidase (GO) Buffer pH 5.5, 50mM Glucose, 300uM substrate, 5uM PHM Quench entire reaction with TFA RPHPLC equipped with Fluorescence Detector Peroxide Concentration Assay GO addition 45g/mL Monitor Oxygen Consumption
- 42. Peroxide Generation by Glucose/Glucose Oxidase (GO)
- 43. Peroxide Reaction Stoichiometry
- the GO-free reaction is
- uncoupled
- the reaction of peroxide with
- PHM generates a species
- capable of perpetuating the
- disproportionation reaction
- the GO reaction is highly
- coupled and the rate of
- product formation remained
- constant.
- peroxide reacts with PHM to
- generate product by a
- pathway that does not
- rely on the simple reduction
- to dicopper (I) and
- subsequent reaction with
- dissolved oxygen
- 44. PHM Kinetics and Thermodynamics
- 45. PHM Kinetics and Thermodynamics
- 46. PHM Kinetics and Thermodynamics
- Why is the peroxide reaction slower?
- Substrate K m of the peroxide vs. ascorbate reaction suggests
- that the substrate is binding to a different form of the enzyme in
- peroxide reaction, perhaps an oxidized form.
- The large increase in K D upon reduction of the enzyme is consistent
- with this theory.
- 47.
- peroxide is not acting as a simple reductant
- peroxide is generating a reactive oxygen species in the cavity
- an intermediate must exist which is equivalent to Cu(I or II)-O 2
-
- Cu(II)-OOH in equlibrium with Cu(I)-O 2
-
-
- requires a reversible ET from Cu H to Cu M
-
-
- Cu(II)-superoxo
-
-
- does not require long range ET
-
- CuH (H172A) and CuM (H242A) deletion mutants showed no activity
- 48. Proposed Mechanism
- 49.
- Peroxide reduces 25% of the Cu centers
- 25% of total Cu(II) was reduced to Cu(I)
-
- independent of incubation time
-
- consistent with mechanistic requirement of Cu H reduction
- 50. Conclusions
- Peroxide is not the intermediate for product formation
- Both ascorbate and peroxide pathways share a common intermediate
- The active intermediate is likely to be a Cu(II)-superoxide
- The entire reaction is taking place inside the active site cavity
- This chemistry provided a foundation for future work
-
-
- Spectroscopic characterization of intermediates
-
-
-
-
- stopped flow and freeze quench techniques combined
-
-
-
-
-
- with UV-Vis, EXAFS, EPR, and FTIR spectroscopy.
-
-
- 51. Exploring the Preference for Met Coordination at CuM
- mutagenesis studies have shown the Met plays a critical role in catalysis
- EXAFS shows that in the oxidized form the CuM site coordinates 2 histidines
- and 2 water molecules in the equatorial plane
- Met is not visible, but is believed to coordinate in the axial plane
- upon reduction the water ligands are displaced as the Met moves closer
- determining the pH dependent correlation between PHM activity, equilibrium
- constants, and structural changes is important for elucidating the role of
- Met in catalysis
- pH-activity profiles and equilibrium constants were determined in Sulfonic
- Acid, (MES/HEPES/CHES) formate/sulfonic-acid, and acetate/sulfonic-acid
- buffer systems (formate or acetate/MES/HEPES/CHES)
- 52. XAS Edge Results from Core Ionization Energies (keV)
- 53. EXAFS Photoelectron Scattering a s E 0 absorption coefficient Energy (eV) 1 E a s 2 E
- 54. Questions XAS Can Address
- What types of atoms are in the first coordination sphere of a metal site ?
- What is the molecular symmetry of this metal site ?
- How covalent are the metal ligand bonds ?
- Does a particular treatment ...
-
- generate a redox change at this metal site?
-
- result in a structural change at this metal site?
- Is this metal part of a metal cluster ?
- 55. Essential Information from EXAFS How many of what type of ligands are at what distance from metal? Observable Frequency Phase Shift Amplitude Information Distance Type of Atom # of Atoms
- 56. EXAFS of Oxidized PHM Shell R( ) 2 2 ( -1 ) 2.5 N(im) 1.97 0.009 1.5 O/N 1.97 0.009 Peaks at ~2 (Cu-N/O) ~ 3 (C2/C5 imidazole) ~ 4 (C3/N4 imidazole) Cu N1 C2 C5 N4 C3 Cu N1 C2 C5 N4 C3
- 57. EXAFS of the reduced PHM shows major changes in coordination First shell is split into two peaks at ~1.90 (Cu-N) and ~2.3 (Cu-S) Outer shell signatures of histidine are still present Histidine shell splits if copper sites are refined separately Shell R() 2 2 ( -1 ) 1.0 N(im) 1.98 0.007 0.5 S(met) 2.26 0.003 1.0 N(im) 1.88 0.007
- 58. pH-activity profiles Acetate system Sulfonic Acid system
-
- shifted the pH maximum from 5.8 to 7.0
-
- active species forms at 5.8 and decays at 8.3
-
- exhibited a pH maximum of 5.8
-
- inactive at pH > 9 (borate)
- broad maximum from pH 5.5 to 6.0 then declined
-
- 59.
-
- a single active species with pKas of 6.8
-
- and 8.2
-
- a protonated unreactive species A
-
- a major reactive species B formed at pKa
-
- 4.6
-
- a less reactive C with pKas of 6.8 and 8.2
- The formate system fits to:
- a protonated unreactive species A
- a reactive species B with pKas of 4.7 and 6.8.
- The acetate system fits to:
- The sulfonic acid system fits to:
-
- 60.
-
-
- apparent K m of substrate decreased from pH 5-8
-
-
-
- K d did not decrease with pH, but varied with oxidation state
-
-
-
-
- change in apparent Km is likely due to a shift to reduction as the rate
-
-
-
-
- determining step (zero-order for substrate)
-
-
-
-
- rate dominated by K cat
-
-
-
- 61. Significance of pH Rate Data
- determined pH dependence of other markers in both oxidative states
- and correlated them to the pH rate data
-
- EPR and XAS
-
-
- XAS simulation give rise to a number of paramaters including
-
-
-
- coordination distances, numbers, and ligand identity
-
-
-
- DW factor
-
-
-
-
- measures attenuation of X-ray scattering from thermal motion
-
-
-
-
-
- or quenched disorder
-
-
-
-
- absorption edges (8983 eV)
-
-
-
-
- gives insight into coordination number and oxidation state
-
-
- 62. Acetate System
- oxidized system shows no
- significant changes
- Cu-S (Met) component is intense at
- pH 4.0. and dominates the first shell
- slowly disappears as pH rises
- 63.
- DW factor changes from 22 0.001 2 to 22 0.012 2 in the acetate system
-
- characteristic of a transition to a weakly bound state
-
-
-
-
- Cu-S DW factor changed from 22 0.008 2 to 22 0.012 2
-
-
-
- Simulations which changed copper occupancy were inferior
- 64.
- 8983 eV absorption edge feature
- increases and moves to slightly
- higher energy as the pH increases
- tracks pH transition of Cu-S DW
- indicates a change to a lower
- coordination number
- 65. pH dependence of the Cu-S Debye-Waller Factor
- Both systems show the DW factor to be modulated by a deprotonation
- event, with the pKa of the sulfonic acid system downshifted by ~ 1 pH unit
-
- the acetate system has a pKa of 5.9 .13
-
- the sulfonic acid system has a pKa of 4.8 .10
- 66. Significance of the pH-dependent Data
- Enzyme exists in two forms, Met on and Met off
-
-
-
- pKas for the met off transition are identical to those of formation of the
-
-
-
-
-
- active species
-
-
-
-
-
- Met off form is the active form
-
-
-
- the met off state is a flexible conformer with dynamic disorder along the
-
- Cu-S vector
-
-
-
- tunneling requires conformational mobility
-
-
- 67.
- Is the conformational change localized or
- global?
- Oxidized PHM was photoreduced in the
- X-ray beam at pH 5.1 and 100 K in the
- acetate buffer system.
- isosbestic point indicates formation of
- a single species of reduced enzyme.
- simulation reveals the Met off form and
- that scatterers present in the oxidized
- form have dissociated
- So, although localized changes can
- occur in the frozen matrix, the Met off
- form suggests that the Cu-S transition
- requires changes in more global
- elements.
- Edges at 0, 30, 60, 90, 180 minutes (bottom to top)
- Photoreduced in red, ascorbate reduced in black
- 68.
- It is likely that the Cu_S(Met) transition affects catalysis by participating in
- H-tunneling.
-
- Cu-S(Met) likely samples the same protein dynamics as the
-
- tunneling process
-
- conformational mobility of the substrate relative to the active
-
- copper-superoxo species may allow it to modulate the tunneling probability
-
- by sampling vibrational modes along the Cu-O----H---C coordinate
-
-
- substrate cross-links two beta strands via R240
-
-
-
-
- connected to strand with H242 and H244
-
-
-
-
-
- also connected to strand containing M314 via Y318 and N316
-
-
-
-
-
- C315 anchors the latter strand to C293
-
-
-
- Cu-S(Met) interaction may be transmitted via the substrate-binding
-
- beta strands about the C315 anchor modulating the Cu-O----H---C distance
-
- Back donation of electrons from the weakly bound Met-S may stabalize the
-
- Cu(I) form, increasing the probability of tunneling by increasing the driving
-
- force
- 69. Present Work
- characterization of complete S transition with the formate buffer system
- structural and kinetic characterization of M314H
- characterization of redox kinetics using stopped flow and freeze quench
- techniques in conjunction with EPR and XAS.
- kinetic and structural characterization of PHM activators and inhibitors
- 70. Future Work
- One experiment too many
- M314H EXAFS revealed that although M314 is critical for catalysis, it
- is not responsible for the on/off transition
-
- identify the source of the S signal
-
- reexamine oxygen binding preferences
-
- reexamine the role of M314
- Characterize the active oxygen intermediate by using mutants and
- slow substrates to cause it to accumulate in the active site cleft
- Determine viability of ET pathways using a photoactivatable reductant
- TUPS (thiouredo-pyrenesulfonate)
-
- substrate bound TUPS for the substrate mediated pathway
-
- bind TUPS to residues with short pathways to the Cu centers
- 71. Acknowledgements NIH DOE Stanford Synchotron Radiation Laboratory Staff Ninian Blackburn, Ph.D. Pierre Moienne Loccoez, Ph.D. Caitlin Grammer Gnana Sutha, Ph.D. Martina Ralle, Ph.D. Luisa Andruzzi, Ph.D. Joel Burchfiel
- 72.
- 73.
- 74.
- 75.
- 76.
- 77.
- 78.
- 79.