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 s at 2C 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 UC-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 UC-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-ethylmaleimide. The reactive sulfhydryl groups are likely to be those generated by reduction of a disulfide during activation. 20 references, 3 figures, 2 tables.
- Research Organization:
- Oak Ridge National Lab., TN
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 6297749
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Vol. 236:2
- Country of Publication:
- United States
- Language:
- English
Similar Records
Interaction of 5'-P-sulfonylbenzoyl adenosine with cysteine residues of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
Identification of cysteine-319 as the target amino acid of 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5 prime -triphosphate in bovine liver glutamate dehydrogenase
Related Subjects
CYSTEINE
ALKYLATION
NUCLEOTIDES
RECEPTORS
PHOSPHOTRANSFERASES
ENZYME ACTIVITY
ADP
ATP
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
COVALENCE
EXPERIMENTAL DATA
PHOSPHATES
REAGENTS
SPINACH
TRACER TECHNIQUES
AMINO ACIDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DATA
ENZYMES
FOOD
INFORMATION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PLANTS
REACTION KINETICS
THIOLS
TRANSFERASES
VEGETABLES
550201* - Biochemistry- Tracer Techniques