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), Journal Name: Biochemistry; (United States) Vol. 26:6; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ADENOSINE
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
CHEMICAL REACTIONS
CHEMISTRY
DECOMPOSITION
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
HYDROLYSIS
INACTIVATION
KINETICS
LABELLED COMPOUNDS
LABELLING
LYSIS
MICROORGANISMS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC FLUORINE COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
REACTION KINETICS
RIBOSIDES
SOLVOLYSIS
STOICHIOMETRY
SULFONATES
TRANSFERASES
TRITIUM COMPOUNDS