Fast and slow cyclic nucleotide-dissociation sites in cAMP-dependent protein kinase are transposed in type Ibeta cGMP-dependent protein kinase.
Both cyclic GMP-dependent protein kinase (cGK) and cyclic AMP-dependent protein kinase (cAK) contain two distinct cyclic nucleotide-binding sites referred to as fast and slow sites based on cyclic nucleotide dissociation behavior. In cAK, the fast site lies amino-terminal to the slow site, and sequence homologies between cAK and cGK have suggested similar positioning for the sites in cGK. Recombinant human type Ibeta cGK (wild type (WT) cGK) was overexpressed, and the properties of purified WT cGK and native type Ibeta cGK were similar. cGK was mutated singly at Thr-193 (T193A, T193V, and T193S) and Thr-317 (T317A, T317V, and T317S), which have been predicted to provide cGMP specificity in the cGMP-binding sites of cGK; a double mutant (T193A/T317A) was produced also. Compared with WT cGK, half-maximal activation (Ka) of mutant cGKs by cGMP was increased 2- (T317A), 27- (T193A), or 63-fold (T193A/T317A), but the Ka for cAMP of these mutants was essentially unchanged. The T193A and T193V mutants had a large increase in the rate of the slow component of [3H]cGMP dissociation, but in the T317A and T317V mutants, there was no change in the slow component. The T193S and T317S mutants had only minor effects on [3H]cGMP dissociation, thus establishing the importance of the hydroxyl group of Thr-193 and -317 for cGMP binding to cGK. Thus, in type Ibeta cGK, the slow cGMP-binding site is identified as the amino-terminal site in contrast to the order assigned to the fast and slow cAMP-binding sites of cAK.
Reed
R B
RB
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Sandberg
M
M
Jahnsen
T
T
Lohmann
S M
SM
Francis
S H
SH
Corbin
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013912:Threonine; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013912:Threonine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP