Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase.
Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation.
Omnaas
J
J
Porter
M A
MA
Hartman
F C
FC
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004983:Ethanolamines / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology*
D004983:Ethanolamines; D009711:Nucleotides; C020161:N-bromoacetylethanolamine phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine; D005033:Ethylmaleimide