Multiple molecular forms of glutamine synthetase produced by enzyme catalyzed adenylylation and deadenylylation reactions

Abstract

The glutamine synthetase from E. coli has a molecular weight of 600,000 and is composed of 12 apparently identical subunits that are arranged in two superimposed hexagonal layers. The activity of the enzyme is modulated by the covalent attachment of one 5′-adenylyl group to each subunit of the enzyme. Adenylylation is catalyzed by a specific ATP:adenylyltransferase that catalyzes a transfer of the adenylyl moiety from ATP to a particular tyrosyl hydroxyl group of each subunit. Adenylylation is accompanied by a decrease in catalytic potential, conversion from an Mg 2+-dependent to Mn 2+-dependent enzyme, a shift in the pH optimum from 8.0 to 6.9 and an increase in susceptibility to feedback inhibition by CTP, AMP, histidine and tryptophan. Deadenylylation of adenylylated enzyme is catalyzed by a complex system in which two protein fractions participate. The products are unadenylylated enzyme and AMP. Oppositely directed effects of glutamine, α-ketoglutarate and UTP on the activities of the two glutamine synthetase modifying enzymes provide an effective means of regulating glutamine metabolism in response to variations in the availability of ammonia nitrogen. Glutamine activates the adenylyltransferase but it is a potent inhibitor of the deadenylylating enzyme system. On the other hand, α-ketoglutarate and UTP are required for the activity of the deadenylylating enzyme but they inhibit the adenylyltransferase. High levels of ammonium salts favor the conversion of α-ketoglutarate to glutamine, but when the level of ammonium salt is low, α-ketoglutarate will tend to accumulate. Therefore, the presence of high levels of ammonium salts favors the conversion of α-ketoglutarate to glutamine, but when the level of ammonium salt is low, α-ketoglutarate will tend to accumulate. Therefore, in the presence of high levels of ammonium nitrogen, the resultant high ratio of glutamine to α-ketoglutarate will facilitate conversion of unadenylylated glutamine synthetase to a less active Mn 2+-dependent form. However, when the supply of ammonium nitrogen is low the glutamine/α-ketoglutarate ratio will also be low and will favor deadenylylation (activation) of the glutamine synthesis. In addition to their fine control of glutamine synthetase activity, the reciprocal effects of α-ketoglutarate, glutamine and UTP on the two modifying enzymes avoids a useless, undirected coupling of their activities, which would result in wasteful hydrolysis of ATP to AMP and PPi. Considering the fact that glutamine synthetase is composed of 12 apparently identical subunits, each one of which can be adenylylated, it has been calculated by M. S. Raff and W. C. Blackwelder that 382 molecular forms of glutamine synthetase are possible, each differing with respect to the number and relative orientation of the adenylylated subunits within the same molecules; i.e., within hybrid molecules. Evidence has been obtained indicating that hybrid molecules do exist and that heterologous interaction between adenylylated and unadenylylated subnunits in such hybrid molecules affect the molecular stability, and the biosynthetic catalytic potential of both kinds of subunits. In addition these interactions affect the apparent affinity of the enzyme for the substrate L-glutamate. Studies of the γ-glutamyltransferase activity of various forms of glutamine synthetase indicate that the transferase activity of each kind of subunit in hybrid molecules is expressed independently of each other. In other words, heterologous subunit interaction effects are uncoupled in so far as the transferase catalytic potential is concerned. By taking advantage of the fact that at pH 7.15 the transferase activity of both kinds of subunits is the same, and the further fact that in the presence of Mg 2+ only unadenylylated subunits are active, a method was developed which permits the determination of the average state of adenylylation of glutamine synthetase, even in relatively impure preparations.