Interaction of 5'-P-sulfonylbenzoyl adenosine with cysteine residues of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6074897

El-Maghrabi, M R; Lively, M O; Pilkis, S J

The kinase and bisphosphatase reactions of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase appear to be catalyzed at separate active sites. The kinase site contains 3 cysteinyl residues that are important for sugar phosphate binding but not for ATP binding. These groups are readily alkylated with iodoacetamide which decreases by 15-fold the affinity for Fru 6-P but also increases the maximal velocity of the reaction by the same extent. Incubation of the enzyme with 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP analog, has no effect on the bisphosphatase activity but inactivates the kinase. The addition of dithiothreitol completely reactivates the kinase, suggesting that the reagent affected sulfhydryl groups critical for sugar phosphate binding and not the ATP site of the enzyme. Similarly, 8-Azido-ATP/UV-photoinactivated enzyme is also reactivated by dithiothreitol and involves the same sulfhydryl groups, since alkylation of the latter with iodoacetamide protects the enzyme from inactivation by FSBA and from 8-azido ATP. Cyanogen bromide cleavage of enzyme that had been alkylated with iodo(I-/sup 14/C)acetamide yielded a 20,000 dalton peptide which contained the three cysteinyl residues. It is concluded that the site of action of ATP analogs to inactivate the kinase are these cysteinyl residues rather than the ATP binding site per se.

• Research Organization: State Univ of New York, Stony Brook
• OSTI ID: 6074897
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETAMIDE
ADENOSINE
AMIDES
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
ATP
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CYSTEINE
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYMES
ESTERASES
GLANDS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIVER
MAMMALS
MOLECULAR STRUCTURE
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PHOSPHATASES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
RATS
REACTION KINETICS
RESIDUES
RIBOSIDES
RODENTS
THIOLS
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES