[Frontiers in Bioscience S5, 650-660, January 1, 2013]

Catalytic site amino acids of PKGI-alpha influence allosteric cGMP binding

Jennifer L. Busch1, Thomas M. Bridges2, Robyn Richie-Jannetta3, Brian P. Hollett1, Sharron H. Francis2, Jackie D. Corbin2

1Department of Biology, Wheaton College, Wheaton, IL 60187; 2Department of Molecular Physiology and Biophysics and 3Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-0615

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Site-directed mutagenesis of PKGI-alpha
3.2. Expression of PKGI-alpha protein
3.3. Purification of recombinant PKGI-alpha
3.4. Determination of PKGI-alpha catalytic activity
3.5. Determination of Ka (cGMP and cAMP) values
3.6. Determination of KD for cGMP
3.7. Determination of Km and Vmax for heptapeptide substrates and Km for ATP
3.8. Data analysis
3.9. Materials
4. Results
4.1. Cyclic nucleotide-binding affinities of PKGI-alpha catalytic domain mutants are increased
4.2. Effect of PKGI-alpha catalytic domain mutations on autoinhibition
4.3. Effects of PKGI-alpha catalytic domain mutations on phosphotransferase activity
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (Ka) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (KD) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (Km) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.