5′-Nucleotidyl-O-tyrosine bond in glutamine synthetase

Abstract

This chapter discusses the 5'-adenylyl-O-tyrosine and 5'-uridylyl-O-tyrosine linkage involved in the regulation of glutamine synthetase (GS). GS is composed of 12 identical subunits and plays a central role in the assimilation of ammonia in Escherichia coli . Cellular GS is regulated in response to the quality and abundance of the nitrogen source. One way to regulate GS activity in E. coli involves the cyclic adenylylation and deadenylylation of a unique tyrosine group in each subunit. The adenylylation reaction is catalyzed at the adenylylation site (AT a ) of an adenylyl transferase (AT) and involves the transfer of adenylyl groups from ATP to the enzyme with the concomitant formation of PP i . The removal of the adenylyl group from GS is achieved by the phosphorolysis of the adenylyl- O -tyrosyl bond to yield ADP and unmodified GS. This reaction is catalyzed at the deadenylylation site (AT d ) of AT. Several methods are available to prove the presence of adenylylated GS in E. coli . The chapter describes a procedure used most widely. The procedure requires measuring GS activity in the Mn 2+ -triethanolamine-dimethylglutaric acid (TEA-DMG) buffer, with and without MgCl 2 .