ATP:Glutamine synthetase adenylyltransferase from Escherichia coli: Purification and properties of a low-molecular weight enzyme form

Abstract

Some kinetic properties of a low-molecular weight form of the ATP: glutamine synthetase adenylyltransferase of Escherichia coli are reported. Described also is a purification procedure from which the small adenylyltransferase of ~70,000 molecular weight is purified ~500-fold from E. coli extracts and is derived from a larger active protein (~130,000 molecular weight) that is a component of the deadenylylating enzyme system of this microorganism. The small adenylyltransferase isolated can be purified a further 1.7-fold in polyacrylamide gel electrophoresis. The incorporation of 5′-adenylyl groups into the glutamine synthetase structure is optimal at pH ≥ 7.8, 30 m m MgCl 2 (15 m m MnCl 2 is about one-third as effective an activator as is MgCl 2), 10 m m l-glutamine, and 5 m m ATP. Anions, α-ketoglutarate, UTP, l-glutamate, various divalent cations with MgCl 2 or excess MgCl 2 (MnCl 2), and sulfhydryl reagents are inhibitory. The adenylyltransferase and unadenylylated glutamine synthetase have an apparent association constant of ~5 × 10 5 m −1. With pyrophosphate present, the small adenylyltransferase catalyzes the removal of 5′-adenylyl groups from adenylylated glutamine synthetase, and ATP has some activating effect on this reverse reaction. Dodecameric glutamine synthetase that has been coupled to Sepharose 4B is enzymatically active and has been used as a solidstate substrate for some experiments with the adenylyltransferase. The adenylyltransferase, however, is inactive with subunits of glutamine synthetase that have been reacted with p-chloromercuriphenylsulfonate and attached to Sepharose 4B to prevent their reassociation during the removal of denaturants.