Proximate sulfhydryl groups in the acetylglutamate complex of rat carbamylphosphate synthetase I: Their reaction with the affinity reagent 5′- p-fluorosulfonylbenzoyladenosine

Abstract

A preparation of rat carbamylphosphate synthetase I, isolated in the presence of antipain and stable without glycerol, has been used to investigate the effect of the allosteric activator, N-acetyl- l-glutamate (AcGlu), on the sulfhydryl chemistry of the enzyme. The enzyme · AcGlu complex was rapidly inactivated by several sulfhydryl group reagents and the ATP analog, 5′- p-fluorosulfonylbenzoyladenosine (FSO 2BzAdo), with the loss of two sulfhydryl groups per monomer. Inactivation was much slower without AcGlu, and ATP/Mg 2+/K + provided complete protection. Reaction with a 1.1 molar excess of 4,4′-dipyridyldisulfide resulted in an intramonomer disulfide bond between groups that are probably juxtaposed in the activated enzyme, because 1.1 equivalents of the vicinal dithiol reagent, phenylarsine oxide, eliminated the rapid reaction with the disulfide. Evidence is presented that the same disulfide bond was formed in the reactions with 5-thiocyano-2-nitrobenzoic acid and FSO 2BzAdo. Inactivation by FSO 2BzAdo was a pseudo-first-order reaction. The concentration dependence of the rate is consistent with the reaction proceeding through a noncovalent complex ( K I = 67 μm and k 2 = 0.23 min −1 at pH 7.0, 30 °C). Protection from FSO 2BzAdo by ATP required Mg 2+ in excess of ATP with K MgATP = 4.5 μm at saturating free Mg 2+ (0.1 m K +) and K Mg 2+ = 6.5 m. K MgATP is close to K d for the molecule of ATP that contributes the phosphoryl group of carbamylphosphate (H. B. Britton, V. Rubio, and S. Grisolia, (1979) Eur. J. Biochem. 102, 521–530]; K Mg 2+ agrees with the minimum value for the steady-state kinetic parameter, K i,Mg 2+ , obtained under the same conditions. Dissociation constants for adenosine (320 μ m), MgADP (110 μ m) at 10 m m Mg 2+, and AcGlu (100 μ m) were also estimated.