Inactivation of Escherichia coli glycerol kinase by 5'-(p-(fluorosulfonyl)benzoyl))adenosine: protection by the hydrolyzed reagent
Incubation of Escherichia coli glycerol kinase with 5'-(p-(fluorosulfonyl)benzoyl)adenosine (FSO/sub 2/BzAdo) at pH 8.0 and 25/sup 0/C results in the loss of enzyme activity, which is not restored by the addition of ..beta..-mercaptoethanol or dithiothreitol. The FSO/sub 2/BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis. The hydrolysis of the reagent has two effects on the observed kinetics. The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent. The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product. Determinations of the reaction stoichiometry with /sup 3/H-labeled FSO/sub 2/BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit. Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO/sub 2/BzAdo provide protection from inactivation. The protection obtained with ATMP is not dependent on Mg/sup 2 +/. The results are consistent with modification by FSO/sub 2/BzAdo of a single adenine nucleotide binding site per enzyme subunit.
- Research Organization:
- Texas A and M Univ., College Station
- OSTI ID:
- 6470480
- Journal Information:
- Biochemistry; (United States), Vol. 26:6
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ADENOSINE
LABELLING
PHOSPHOTRANSFERASES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ESCHERICHIA COLI
INACTIVATION
ORGANIC FLUORINE COMPOUNDS
STOICHIOMETRY
SULFONATES
TRITIUM COMPOUNDS
BACTERIA
CHEMICAL REACTIONS
CHEMISTRY
DECOMPOSITION
ENZYMES
HYDROLYSIS
KINETICS
LABELLED COMPOUNDS
LYSIS
MICROORGANISMS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
REACTION KINETICS
RIBOSIDES
SOLVOLYSIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques