Affinity labeling of rat liver carbamyl phosphate synthetase I by 5'-p-fluorosulfonylbenzoyladenosine.
The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been used to study the interaction of MgATP with rat liver carbamyl phosphate synthetase I. Incubation of the enzyme with concentrations of FSBA as low as 0.025 mM produced considerable inactivation (41% at 120 min); identical rates and extents of reaction were produced by 0.5, 1, and 2 mM FSBA. Of the substrates for carbamyl phosphate synthetase I, only MgATP protected against FSBA inactivation. In the presence of a constant concentration of MgATP, increasing the FSBA concentration led to increased inhibition. Conversely, an increase in MgATP concentration led to decreased inhibition from a constant concentration of FSBA. Other nucleotide triphosphates provided no protection against FSBA inactivation. Addition of dithiothreitol to the FSBA-inactivated enzyme led to partial reactivation, suggesting that cysteine residue(s) were involved in the FSBA reaction. 5,5'-Dithiobis(2-nitrobenzoic acid) titration of the free sulfhydryl groups on the enzyme confirmed that cysteine residues were involved in reaction with FSBA; titration of the enzyme after incubation in the absence and presence of FSBA yielded values of 21 and 18(+/- 1), respectively. Binding studies with 5'-p-fluorosulfonylbenzoyl[2-3H]adenosine indicated that: 4 amino acid residues were involved in reaction with FSBA; 2 of these reaction sites were cysteine residues and 2 were noncysteine residues; MgATP protected one of the cysteine residues and one of the noncysteine residues from reaction with FSBA; the MgATP-protected noncysteine residue is essential for fully activity. These data strongly suggest that FSBA is an affinity label for two distinct MgATP sites on carbamyl phosphate synthetase I.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D051381:Rats; D013329:Structure-Activity Relationship
D000345:Affinity Labels; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D000241:Adenosine