MgATP-induced conformational change of the catalytic subunit of cAMP-dependent protein kinase.
Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of gamma-phosphate transfer from adenosine 5'-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg(2+), ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit ((CF)C). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times phi(F) and phi(S). The (CF)C apoenzyme exhibited the rotational correlation times phi(F)=1.8+/-0.3 ns and phi(S)=20.1+/-0.6 ns which were reduced to phi(F)=1.1+/-0.2 ns and phi(S)=13.3+/-0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the (CF)C subunit adopted a more compact shape upon formation of a (CF)C.MgATP binary complex. Neither Mg(2+) (1-3 mM) nor ATP (0.4 mM) alone induced changes in the (CF)C subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the (CF)C x MgATP x IP20 ternary complex produced rotational correlation times similar to those of the (CF)C x MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation.
Yang
Shumei
S
Department of Chemistry, California State University, San Bernardino, CA 92407, USA. syang@csusb.edu
Rogers
Kestrel M
KM
Johnson
David A
DA
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D002250:Carbon Radioisotopes; D020134:Catalytic Domain; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002250:Carbon Radioisotopes; D004791:Enzyme Inhibitors; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D017868:Cyclic AMP-Dependent Protein Kinases