Arginine Kinase

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Arginine Kinase

• Still learning how enzymes work

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Phosphagen Kinases Arginine Kinase, Creatine
KinasePhysiological role
Cr Mg2ATP4- ? CrPO32- Mg2ADP3- H

• ATP Cellular Energy Currency
• Temporal buffering
• Short term energy homeostasis
• Bursts of cellular activity
• Sprinting
• Heart muscle neurons

E
Log time
sec? min? hr?
ATP Cr Anaerobic Aerobic
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Arginine kinase reaction

• Reversible reaction
• Transition state
• g-phosphoryl presumed trigonal

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Transition state structures

• Several dozen known
• Unimolecular pseudo-unimolecular rxns
• Bimolecular reactions
• Bisubstrate complexes
• Substrate analogs that are covalently joined
• Substrate cofactor
• Isocitrate dehydrogenase dihydrofolate
  reductase
• No representatives of group transfer rxns

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Why phosphagen kinases, creatine kinase?

• History
• Wallace Cleland
• Bill Jencks
• Mildred Cohn
• Jeremy Knowles
• George Kenyon
• Well characterized
• Classical enzymology

• Creatine kinase structure proved difficult
• 7 crystallizations
• 1st structure Kabsch 1996
• 3 Å active site disordered

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Arginine kinase

• Mammals
• Creatine kinase
• Dimer or Octamer
• Arthropods
• Arginine kinase
• 40 sequence identity
• Predominantly monomer

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Expression ? Crystallization

• cDNA clone (Strong Ellington)
• Protein expression (Zhou et al. Chapman)
• PET vector / E. coli
• Mostly inclusion bodies
• Denatured ? purified ? refolded ? further
  purified
• Purified yield 30 mg/L
• Structure revealed 4 amplification errors
• E103Q D112G G116A K351R
• Remote from active site
• do not affect kinetics
• Reverted w.-t. never crystallized!

Strong, S. J., and Ellington, W. R. (1996) Comp.
Biochem. Physiol. 113B, 809-16. Zhou, G.,
Parthasarathy, G., Somasundaram, T., Ables, A.,
Roy, L., Strong, S. J., Ellington, W. R., and
Chapman, M. S. (1997) Protein Science 6, 444-9.
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Transition State Analog

• Transition state g-phosphoryl presumed trigonal
• Nitrogen replaces phosphorus ? unreactive
• Dead-end inhibitory complex
• Distances 20 covalent P 100 non-bonded N

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Crystallography transition state 1.9 Å
2Fo-Fc omit
Zhou, G., Somasundaram, T., Blanc, E.,
Parthasarathy, G., Ellington, W. R., and Chapman,
M. S. (1998) Proc. Nat. Acad. Sci., 95, 8449-54.
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Anatomy of Arginine Kinase

• C-terminal domain
• Residues 115 - 357
• Anti-parallel b-sheet
• 8-stranded
• Flanked by 7 a-helices
• Nucleotide-binding
• Glutamine synthetase
• N-terminal domain
• Residues 1 95
• Flexible linker 96 - 114

1M80
Yousef, M. S., Clark, S. A., Pruett, P. K.,
Somasundaram, T., Ellington, W. R., and Chapman,
M. S. (2003) Protein Sci 12, 103-11..
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Refinement 1.2 Å
2Fo-Fc
Yousef, M. S., Fabiola, F., Gattis, J.,
Somasundaram, T., and Chapman, M. S. (2002) Acta
Crystallogr. D 58, 2009-2017.
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Transition State Structure

• Transition state analog
• ADP NO3 Arginine
• Cleft bridging 2 domains
• Specificity loops

Yousef, M. S., Fabiola, F., Gattis, J.,
Somasundaram, T., and Chapman, M. S. (2002) Acta
Crystallogr. D 58, 2009-2017.
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How is the reaction catalyzed?Initial focus on
the phosphagen sub-site.

• Classical enzymology ? Histidine general base
• Dixon (pH) analysis
• Cook, Kenyon Cleland, 1981
• Chemical modification
• NMR

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Guanidinium sub-site

• What promotes new bond from Nh1 to P??
• What no histidines?
• Glutamates?
• Not the expected pK

Zhou, G., Somasundaram, T., Blanc, E.,
Parthasarathy, G., Ellington, W. R., and Chapman,
M. S. (1998) Proc. Nat. Acad. Sci., 95, 8449-54.
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Rethinking the sequence of events

• Conventional wisdom
• Proton removed from Nh2.
• Then lone-pair attack on P?.
• Isoergonic proton transfer requires high pK base
• New possibility
• Lone pair attack before proton abstraction
• Quaternary Nh2 would be acidic
• Isoergonic base would be Glu/Asp
• Structural evidence?
• Oe314, Oe225 consistent w/ sp3 Nh2.
• Suggest optimizing tetrahedral TS not trigonal

Zhou, G., Somasundaram, T., Blanc, E.,
Parthasarathy, G., Ellington, W. R., and Chapman,
M. S. (1998) Proc. Nat. Acad. Sci., 95, 8449-54.
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Two active site glutamates - Glu225 / Glu314

• Both salt-bridge to substrate
• E225 fully conserved
• E314 only partially conserved
• Not in creatine kinases
• Is either catalytic?
• Creatine kinase mutants implicate homolog of E225
• Eder et al., Wallimann, 2000
• Do both contribute in AK?

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Arginine kinase mutation - Glu225 / Glu314

• E225A suggests E225 essential, but
• Challenges illustrated by E314
• Chimera incl. E314V
• Search ? E225 mutants w/ 1 activity
• Conclusion E225 assists catalysis, not essential

E314D
E225Q
Pruett, P. S., Azzi, A., Clark, S. A., Yousef,
M., Gattis, J. L., Somasundaram, T., Ellington,
W. R., and Chapman, M. S. (2003) J Biol Chem 29,
26952-7.
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Structures E314D E225Q

• E314D
• Not catalytic
• Reduced activity
• Alignment of substrates?
• E225Q
• Catalytic (accessory?)
• Catalytic base?
• Alignment of substrates?

Pruett, P. S., Azzi, A., Clark, S. A., Yousef,
M., Gattis, J. L., Somasundaram, T., Ellington,
W. R., and Chapman, M. S. (2003) J Biol Chem 29,
26952-7.
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Essential Cysteine CK History

• Catalytically Essential
• Covalent modification
• Watts al., (1962 )
• Non-catalytic
• Covalent modification - methylation
• CK - Kenyon al., (1977 )
• Was methylation complete?
• Conformational change - mutagenesis
• Kenyon, Wallimann al. (1993 )
• Mutations affect substrate-binding Synergy
• Low pK (thiolate)
• Spectroscopy Cl?-rescue of some mutants

Furter, R., Furter-Graves, E. M., and Wallimann,
T. (1993) Biochemistry 32, 7022-7029. Wang, P.,
McLeish, M., Kneen, M., Lee, G. Kenyon, G.
(2001) Biochem. 40, 11698.
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C271A structure

• Protein isomorphous w/ WT
• Not determinant of conformational change

largest
Gattis, J., Ruben, E., Fenley, M., Ellington, W.
R., Chapman, M. (2004) Biochem. 43 8680.
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Cysteine mutants kinetics structure
Gattis, J., Ruben, E., Fenley, M., Ellington, W.
R., Chapman, M. (2004) Biochem. 43 8680.
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Possible roles of C271

• Not conformational change
• Catalytic
• Accessory rather than essential
• Some mutations have 1 activity
• Possible accessory roles
• Electrostatic stabilization
• Catalytic base, abstracting proton from distal
  guanidinium nitrogen
• Both supported by QM calculations

Gattis, J., Ruben, E., Fenley, M., Ellington, W.
R., Chapman, M. (2004) Biochem. 43 8680.
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Quantum simulations

• Several catalytic mechanisms
• Pleiotropic roles of several residues
• Deconvolution through computer simulation
• Check computation vs. kinetics of mutants etc..
• QM/MM
• Quantum simulation bond formation / cleavage
• Molecular mechanics for long-range protein
  motions
• Start with QM calibration
• Force-field terms Uncatalyzed reaction

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Nature of high energy bonds

• Nucleotides - conventional wisdom
• Charge repulsion
• Negatively charged phosphates
• Solvation lower energy after hydrolysis
• Opposing resonance
• 2 phosphates compete for same lone pair on
  bridging O
• Lower energy when hydrolyzed

• Relevance to NP bond in phosphagens
• Charge repulsion
• Arginine and phosphate are oppositely charged
• Solvation could be a factor again
• Opposing resonance
• Cited as explanation
• Question of magnitude

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Computational approach

• Quantum calculations of increasing complexity
• Incl. B3LYP/6-311G(d,p) and MP2/6-311G(d,p)
• PCM continuum solvent
• Applied to 9 distinct protonations at 5 sites
• NBO natural bond orbital analysis

Ruben, E.A., Chapman, M.S., and Evanseck, J.D.
2006. J Am Chem Soc in press.
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Opposing resonance in guanidinium phosphates?

• Phosphate protonation affects resonance
• Examine correlation of NP length w/ resonance
• Little correlation resonance unequal
• Opposing resonance not dominant factor

Ruben, E.A., Chapman, M.S., and Evanseck, J.D.
2006. J Am Chem Soc in press.
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Anomeric effect

• n(O) ? s(NP)
• Bond length correlates w/ energy of this
  interaction
• LpXAY
• Anomeric effect
• Major contributor to bond lability
• Also in ATP?
• Catalysis through anomeric effect?

Ruben, E.A., Chapman, M.S., and Evanseck, J.D.
2006. J Am Chem Soc in press in prep.
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MechanismS of catalysis

• General base E225
• Electrostatic / base C271
• Anomeric effect?
• Approximation
• Substrates aligned w/in 4º of optimal

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Evidence supporting alignment in catalysis

• Explains reduced-activity mutants
• Eg. E314D
• Explains unreactive substrate analogs
• Imino-ethyl ornithine D-Arg Citrulline
  Ornithine
• Identified catalytic residues do not yet account
  for full catalytic effect

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Mis-match complex Creatine bound to Arginine
kinase

• (Fits!)
• Specificity not lock--key
• Protein structure the same
• As transition state complex
• Specificity not induced fit
• V314E (CK ? AK) ?
• 38º rotation
• Disordered (3 x higher Bs)
• Why is creatine not a substrate of arginine
  kinase?
• Precise guanidinium alignment required?

Azzi, A., Clark, S. A., Ellington, W. R., and
Chapman, M. S. (2004) Protein Sci 13, 575-85.
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Importance of Approximation in Multi-substrate
Reactions?

• How much greater?
• 1970s Koshland vs. Jencks
• 3rd Millenium Bruice vs. Warshel
• Visualization difficult
• Class 1 enzymes oxidoreductases
• Perturbed Isocitrate DH Mesecar et al., 1997
• Bruice redox cofactors not typical substrates
• Class 2 enzymes transferases
• Bisubstrate complexes
• 2 substrates covalently joined
• Prejudges alignment
• Arginine kinase 1st high resolution structure

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Questions of alignment

• How much of the catalytic effect?
• QM/MM potential of mean force (PMF) calculations

• How is precise alignment achieved
• Dynamically?
• Crystallography of complexes
• NMR to measure ms/µs dynamics

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Crystallography substrate-free AK
2Fo-Fc omit
Yousef, M. S., Clark, S. A., Pruett, P. K.,
Somasundaram, T., Ellington, W. R., and Chapman,
M. S. (2003) Protein Sci 12, 103-11..
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Comparing substrate-free TSA forms

• Domain rotations loop closure
• Close active site
• Remove solvent
• Specificity?
• Dynamic domains
• Four
• Not fully contiguous

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Anisotropic Displacement Analysis1.2 Å
Transition State Complex

• Fit thermal ellipsoids
• Magnitude/direction of motion
• Then model motion to extent possible by
• Translation, Libration, Screw
• Quasi rigid groups atoms w/ correlated motions
• Account for most of motion
• Definition of rigid groups
• Delta matrix analysis
• 4 regions for AK - Similar to dynamic domains
• Implications
• Slow large induced changes correlated to fast
  harmonic motions
• Libration dominates anisotropic motion

Yousef, M. S., Fabiola, F., Gattis, J.,
Somasundaram, T., and Chapman, M. S. (2002) Acta
Crystallogr. D 58, 2009-2017.
36
Alignment is a dynamic process in Induced fit
enzymes

• Crystallography
• Amplitudes
• Frequencies
• NMR
• Amplitude of motion
• Time constants (frequencies) per residue

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NMR Backbone Resonance Assignment

• Sample preparation
• Solubility 75mg/mL
• Isotope enrichment
• 15N, 13C, 2H minimal media
• Exchange unfold/refold

• Foundation for NMR
• Large 42kDa

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Backbone Assignment
Davulcu, O., Clark, S.A., Chapman, M.S., and
Skalicky, J.J. 2005. J Biomol NMR 32 178.
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Chemical shift titrations w/ substrates

• Fast exchange
• gradual shift of resonances
• Follow effects conformational changes?
• Binding constants
• Regions impacted by each substrate

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Substrates chemical shift
?Phosphoarginine Arginine??ATP ADP?
?PhosphoarginineADP?
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Chemical exchange rates

• Relaxation dispersion measurements
• Rex ms/µs range
• Commensurate w/ turnover
• Substrate-free (so far)
• 40 amino acids w/ rates in range
• Predominantly 190 loop
• Dissect dynamics critical to binding, chemical
  steps dissociation

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Conclusions

• Catalysis can be complicated multiple
  mechanisms
• Often overlooked
• Need improved methods to quantify
• Alignment may be important in two-substrate
  reactions
• Alignment can only be achieved dynamically
  through protein conformational change
• Dynamics critical to catalysis ( specificity?)
• Dynamics can be probed w/ NMR relaxation
  dispersion

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Credits

• Students / Post-docs.
• Genfa Zhou
• T. Somasundaram
• Mohammad Yousef
• Pam Pruett
• Jim Gattis
• Jeff Bush
• Arezki Azzi
• Shawn Clark
• Eliza Ruben
• Omar Davulcu
• Nancy Meyer
• Collaborators
• Ross Ellington, Biology,FSU
• Jack Skalicky, NMR,Univ. Utah
• Jeff Evanseck,Computing,Duquesne Univ.