Oxidation of cysteines activates cGMP-dependent protein kinase.
The functional significance of the oxidation/reduction state of sulfhydryl groups of cGMP-dependent protein kinase (cGMP kinase) was studied at 30 degrees C using different metal ions as oxidizing agents. Mn2+, Zn2+, Fe2+, Ni2+, and Co2+ failed to activate cGMP kinase, whereas Cu2+, Cu+, Fe3+, Hg2+, and Ag+ activated cGMP kinase by oxidation with an activity ratio (-cGMP/+cGMP) of about 0.7. The activation was not caused by degradation of the enzyme to a cGMP-independent constitutively active form. Reduction of the Cu(2+)-activated and gel-filtered enzyme with dithiothreitol lowered the activity ratio in the absence of cGMP to 0.17. Oxidation did not change the kinetic and binding parameters of cGMP kinase significantly but reduced the number of titratable sulfhydryl groups from 9.5 +/- 0.7 to 6.0 +/- 0.4 cysteines/75-kDa subunit. The free cysteinyl residues of the native and Cu(2+)-oxidized cGMP kinase were labeled with 4-dimethylaminoazobenzene-4'-iodoacetamide or N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Tryptic peptides of the labeled proteins were isolated and sequenced. The cysteinyl residues oxidized by Cu2+ were identified as disulfide bonds between Cys-117 and Cys-195 and Cys-312 and Cys-518, respectively. Cu2+ activation of cGMP kinase was prevented by mild carboxymethylation of the reduced enzyme with iodoacetamide, which apparently modified these four cysteinyl groups. The results show that cGMP kinase is activated by the formation of at least one intrachain disulfide bridge.
Landgraf
W
W
Institut für Pharmakologie und toxikologie, Technische Universität München, Germany.
Regulla
S
S
Meyer
H E
HE
Hofmann
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003300:Copper / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D006157:Guanosine Monophosphate / Q000378:metabolism; D051528:Guanylate Kinases; D008670:Metals / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism
D008670:Metals; D013438:Sulfhydryl Compounds; D003300:Copper; D006157:Guanosine Monophosphate; D009703:Nucleoside-Phosphate Kinase; D051528:Guanylate Kinases; D003545:Cysteine