Inactivation of Escherichia coli glycerol kinase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine: protection by the hydrolyzed reagent.
Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSO2BzAdo) at pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not restored by the addition of beta-mercaptoethanol or dithiothreitol. The FSO2BzAdo 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. The rate constant for the hydrolysis reaction, determined independently from the kinetics of F- release, is 0.021 min-1 under these conditions. Determinations of the reaction stoichiometry with 3H-labeled FSO2BzAdo 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 FSO2BzAdo provide protection from inactivation. The protection obtained with ATP is not dependent on Mg2+. Less protection is obtained with glycerol, GMP, etheno-AMP, and cAMP. No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or fructose 1,6-bisphosphate. The results are consistent with modification by FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*
D000345:Affinity Labels; D009711:Nucleotides; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D010770:Phosphotransferases; D005991:Glycerol Kinase; D000241:Adenosine