skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

Abstract

To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.

Authors:
 [1];  [1];  [1];  [1];  [1];  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1201297
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 290; Journal Issue: 25; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; crystal structure; enzyme mechanism; molecular dynamics; phosphoryl transfer; protein kinase

Citation Formats

Das, Amit, Gerlits, Oksana O., Heller, William T., Kovalevskyi, Andrii Y., Langan, Paul, and Tian, Jianhui. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit. United States: N. p., 2015. Web. doi:10.1074/jbc.M115.643213.
Das, Amit, Gerlits, Oksana O., Heller, William T., Kovalevskyi, Andrii Y., Langan, Paul, & Tian, Jianhui. Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit. United States. doi:10.1074/jbc.M115.643213.
Das, Amit, Gerlits, Oksana O., Heller, William T., Kovalevskyi, Andrii Y., Langan, Paul, and Tian, Jianhui. Fri . "Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit". United States. doi:10.1074/jbc.M115.643213. https://www.osti.gov/servlets/purl/1201297.
@article{osti_1201297,
title = {Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit},
author = {Das, Amit and Gerlits, Oksana O. and Heller, William T. and Kovalevskyi, Andrii Y. and Langan, Paul and Tian, Jianhui},
abstractNote = {To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca2+ cations with Mg2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.},
doi = {10.1074/jbc.M115.643213},
journal = {Journal of Biological Chemistry},
number = 25,
volume = 290,
place = {United States},
year = {2015},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 9 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

The Protein Kinase Complement of the Human Genome
journal, December 2002


Dynamics of cAMP-Dependent Protein Kinase
journal, August 2001

  • Johnson, David A.; Akamine, Pearl; Radzio-Andzelm, Elzbieta
  • Chemical Reviews, Vol. 101, Issue 8
  • DOI: 10.1021/cr000226k

Kinetic and Catalytic Mechanisms of Protein Kinases
journal, August 2001


PKA-I Holoenzyme Structure Reveals a Mechanism for cAMP-Dependent Activation
journal, September 2007


Divalent metal ions influence catalysis and active-site accessibility in the camp-dependent protein kinase
journal, December 1993


Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains
journal, August 1995


Structure and Allostery of the PKA RII  Tetrameric Holoenzyme
journal, February 2012


Dynamic Features of cAMP-dependent Protein Kinase Revealed by Apoenzyme Crystal Structure
journal, March 2003


Metal-Free cAMP-Dependent Protein Kinase Can Catalyze Phosphoryl Transfer
journal, May 2014

  • Gerlits, Oksana; Das, Amit; Keshwani, Malik M.
  • Biochemistry, Vol. 53, Issue 19
  • DOI: 10.1021/bi5000965

Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with magnesium-ATP and peptide inhibitor
journal, March 1993

  • Zheng, Jianhua; Knighton, Daniel R.; Ten Eyck, Lynn F.
  • Biochemistry, Vol. 32, Issue 9
  • DOI: 10.1021/bi00060a005

Detection of Conformational Changes along the Kinetic Pathway of Protein Kinase A Using a Catalytic Trapping Technique
journal, September 1999

  • Shaffer, Jennifer; Adams, Joseph A.
  • Biochemistry, Vol. 38, Issue 37
  • DOI: 10.1021/bi991109q

Dynamics connect substrate recognition to catalysis in protein kinase A
journal, October 2010

  • Masterson, Larry R.; Cheng, Cecilia; Yu, Tao
  • Nature Chemical Biology, Vol. 6, Issue 11
  • DOI: 10.1038/nchembio.452

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy
journal, April 2011

  • Masterson, L. R.; Shi, L.; Metcalfe, E.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 17
  • DOI: 10.1073/pnas.1102701108

Electronic Measurements of Single-Molecule Catalysis by cAMP-Dependent Protein Kinase A
journal, May 2013

  • Sims, Patrick C.; Moody, Issa S.; Choi, Yongki
  • Journal of the American Chemical Society, Vol. 135, Issue 21
  • DOI: 10.1021/ja311604j

Stereochemical course of the phospho group transfer catalyzed by cAMP-dependent protein kinase
journal, April 1988

  • Ho, Mengfei.; Bramson, H. Neal.; Hansen, David E.
  • Journal of the American Chemical Society, Vol. 110, Issue 8
  • DOI: 10.1021/ja00216a068

cAMP-dependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer
journal, February 1994

  • Madhusudan, ; Trafny, Elzbieta A.; Xuong, Nguyen-Huu
  • Protein Science, Vol. 3, Issue 2
  • DOI: 10.1002/pro.5560030203

Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase
journal, March 2002

  • Madhusudan, ; Akamine, Pearl; Xuong, Nguyen-Huu
  • Nature Structural Biology, Vol. 9, Issue 4
  • DOI: 10.1038/nsb780

Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity
journal, June 2012

  • Kovalevsky, Andrey Y.; Johnson, Hanna; Hanson, B. Leif
  • Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 7
  • DOI: 10.1107/S0907444912014886

Role of N-Terminal Myristylation in the Structure and Regulation of cAMP-Dependent Protein Kinase
journal, September 2012

  • Bastidas, Adam C.; Deal, Michael S.; Steichen, Jon M.
  • Journal of Molecular Biology, Vol. 422, Issue 2
  • DOI: 10.1016/j.jmb.2012.05.021

Chemical Basis for Enzyme Catalysis
journal, May 2000

  • Bruice, Thomas C.; Benkovic, Stephen J.
  • Biochemistry, Vol. 39, Issue 21
  • DOI: 10.1021/bi0003689

A View at the Millennium:  the Efficiency of Enzymatic Catalysis
journal, March 2002

  • Bruice, Thomas C.
  • Accounts of Chemical Research, Vol. 35, Issue 3
  • DOI: 10.1021/ar0001665

HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

A short history of SHELX
journal, December 2007

  • Sheldrick, George M.
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 64, Issue 1, p. 112-122
  • DOI: 10.1107/S0108767307043930

Features and development of Coot
journal, March 2010

  • Emsley, P.; Lohkamp, B.; Scott, W. G.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444910007493

2.2 Å refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MnATP and a peptide inhibitor
journal, May 1993

  • Zheng, J.; Trafny, E. A.; Knighton, D. R.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 49, Issue 3
  • DOI: 10.1107/S0907444993000423

GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit
journal, February 2013


Comparison of multiple Amber force fields and development of improved protein backbone parameters
journal, November 2006

  • Hornak, Viktor; Abel, Robert; Okur, Asim
  • Proteins: Structure, Function, and Bioinformatics, Vol. 65, Issue 3
  • DOI: 10.1002/prot.21123

Improved side-chain torsion potentials for the Amber ff99SB protein force field
journal, January 2010

  • Lindorff-Larsen, Kresten; Piana, Stefano; Palmo, Kim
  • Proteins: Structure, Function, and Bioinformatics
  • DOI: 10.1002/prot.22711

NMR-Based Protein Potentials
journal, August 2010

  • Li, Da-Wei; Brüschweiler, Rafael
  • Angewandte Chemie International Edition, Vol. 49, Issue 38
  • DOI: 10.1002/anie.201001898

Phosphoryl Transfer by Protein Kinase A Is Captured in a Crystal Lattice
journal, March 2013

  • Bastidas, Adam C.; Deal, Michael S.; Steichen, Jon M.
  • Journal of the American Chemical Society, Vol. 135, Issue 12
  • DOI: 10.1021/ja312237q

Charge-Balanced Metal Fluoride Complexes for Protein Kinase A with Adenosine Diphosphate and Substrate Peptide SP20
journal, November 2012

  • Jin, Yi; Cliff, Matthew J.; Baxter, Nicola J.
  • Angewandte Chemie International Edition, Vol. 51, Issue 49
  • DOI: 10.1002/anie.201204266

Thiol and amino analogs as alternate substrates for glycerokinase from Candida mycoderma
journal, July 1989


Mechanisms of signaling and related enzymes
journal, December 1997


Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies
journal, June 1987


Is There a Catalytic Base in the Active Site of cAMP-Dependent Protein Kinase? 
journal, March 1997


Phosphorylation Reaction in cAPK Protein Kinase-Free Energy Quantum Mechanical/Molecular Mechanics Simulations
journal, November 2007

  • Valiev, Marat; Yang, Jie; Adams, Joseph A.
  • The Journal of Physical Chemistry B, Vol. 111, Issue 47
  • DOI: 10.1021/jp074853q

Crystal Structure of a cAMP-dependent Protein Kinase Mutant at 1.26 Å: New Insights into the Catalytic Mechanism
journal, February 2004

  • Yang, Jie; Ten Eyck, Lynn F.; Xuong, Nguyen-Huu
  • Journal of Molecular Biology, Vol. 336, Issue 2
  • DOI: 10.1016/j.jmb.2003.11.044

A QM/MM study of Kemptide phosphorylation catalyzed by protein kinase A. The role of Asp166 as a general acid/base catalyst
journal, January 2015

  • Pérez-Gallegos, Ayax; Garcia-Viloca, Mireia; González-Lafont, Àngels
  • Physical Chemistry Chemical Physics, Vol. 17, Issue 5
  • DOI: 10.1039/C4CP03579H

Synchronous Opening and Closing Motions Are Essential for cAMP-Dependent Protein Kinase A Signaling
journal, December 2014

  • Srivastava, Atul K.; McDonald, Leanna R.; Cembran, Alessandro
  • Structure, Vol. 22, Issue 12
  • DOI: 10.1016/j.str.2014.09.010

A Transition Path Ensemble Study Reveals a Linchpin Role for Mg 2+ during Rate-Limiting ADP Release from Protein Kinase A
journal, December 2009

  • Khavrutskii, Ilja V.; Grant, Barry; Taylor, Susan S.
  • Biochemistry, Vol. 48, Issue 48
  • DOI: 10.1021/bi901475g

    Works referencing / citing this record:

    Molecular Fingerprints for a Novel Enzyme Family in Actinobacteria with Glucosamine Kinase Activity
    journal, May 2019

    • Manso, José A.; Nunes-Costa, Daniela; Macedo-Ribeiro, Sandra
    • mBio, Vol. 10, Issue 3
    • DOI: 10.1128/mbio.00239-19

    Molecular Fingerprints for a Novel Enzyme Family in Actinobacteria with Glucosamine Kinase Activity
    journal, May 2019

    • Manso, José A.; Nunes-Costa, Daniela; Macedo-Ribeiro, Sandra
    • mBio, Vol. 10, Issue 3
    • DOI: 10.1128/mbio.00239-19