A presentation of my work on peptidyl-hydroxylating monooxygenase conducted at Oregon Health and Science University.
79
Production and Mechanistic Characterization of Peptidylglycine Hydroxylating Monooxygenase (PHM) Andrew Bauman Senior Research Associate @ OHSU
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
(a) Resting State Blackburn et. al ., J. Biol. Chem. 5 (2000)
341-353. 11 Contact 80 2.25
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
Research Aims
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
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
B1
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
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
18 O 2 Incorporation Experiments H 2 18 O 2 under atmospheric 16 O
2 ( a ), H 2 16 O 2 under atmospheric 18 O 2 ( b ), H 2 18 O 2
under anaerobic conditions c ), and H 2 18 O 2 under atmospheric 18
O 2 ( d ).
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
18 O 2 Incorporation Experiments
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
Experimental Deductions
48. Proposed Mechanism
49.
Peroxide reduces 25% of the Cu centers
EPR Spectrum of Peroxide Treated PHM
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)
MES/HEPES/CHES Acetate/MES/HEPES/CHES
broad maximum from pH 5.5 to 6.0 then declined
Formate System (Formate/MES/HEPES/CHES)
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
Acetate system, ascorbate reduced
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
Acetate system, pH 4.0, 5, 5.5, 6.0 (bottom to top)
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
Conclusions
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