How did glutamine synthetase become a subject of long-term research in Earl's laboratory? Why was it so important to study the various properties of this enzyme? Glutamine synthetase catalyzes the conversion of one amino acid, glutamate, to another, glutamine. Glutamine then serves as an important source of nitrogen in the synthesis of various cellular molecules. By catalyzing the synthesis of glutamine, glutamine synthetase can not only affect the subsequent transformation of glutamine into other amino acids, but its activity is also subject to "feedback inhibition" of seven different end products in the branched pathway of glutamine metabolism. What was striking about glutamine synthetase, Earl and Woolfolk found, was that each of these end products inhibited its activity in a "cumulative fashion." In other words, inhibition was more effective when more end products were involved, and the enzymatic activity was almost completely switched off when all the end products were bound to glutamine synthetase. Earl and Woolfolk referred to this mechanism as "cumulative feedback inhibition."

Earl with Bennett Shapiro and Ann Ginsberg, 1968

In the late 1960s, Earl and his co-workers were faced with a puzzling problem: a new sample of glutamine synthetase, prepared from extracts of E. coli by the same procedure developed for the previous batch, was insensitive to cumulative feedback inhibition. After an intensive, exhaustive search to explain this, they finally found a mechanism by which the attachment of the molecule called AMP (adenosine monophosphate) to—and also its detachment from—specific sites of glutamine synthetase could dramatically change its susceptibility to feedback inhibition. The previous sample then turned out to be AMP-attached glutamine synthetase, which was very responsive to feedback inhibition, whereas the new preparation was the AMP-detached one. The attachment and detachment of AMP, called "adenylylation" and "deadenylylation," took place through the formation and breakdown of a "covalent" bond (a kind of chemical bond) between AMP and a specific part of glutamine synthetase. And this process was catalyzed by the same enzyme, adenylyl transferase . It was thus an example of "reversible covalent modification ." Earl and his co-workers provided additional evidence for this mechanism with detailed studies of the structure of glutamine synthetase, both the active and inactive forms, by electron microscopy.

Ground-breaking research often creates many unanswered questions. If adenylyl transferase can catalyze both adenylylation and deadenylylation processes, what makes this enzyme tilt the equilibrium of reactions in one direction? What regulates its activity? This question was answered by Michael Brown and Amiel Segal, postdoctoral fellows in Earl's laboratory. Working together, Brown and Segal demonstrated that the ability of a regulatory protein to promote adenylylation and deadenylylation of glutamine synthetase was dictated by the activity of another enzyme, uridylyl transferase. This enzyme was later purified and characterized by Sue Goo Rhee in the laboratory. Depending on the cellular concentration of various metabolites, this enzyme can catalyze the covalent attachment of a uridylyl group ( UMP, uridine monophosphate) to a unique tyrosine residue of the regulatory protein and thereby determine its ability to promote adenylylation or deadenylylation of glutamine synthetase. By the mid-1970s, the scientists were convinced that the activity of glutamine synthetase was controlled by a "cascade system," in which the two systems of reversible covalent modification were tightly linked. Each system was composed of two reversible reactions, or what they called two interconvertible enzyme cycles, the linkage of which resulted in the formation of a "bicyclic cascade system."

In the late 1970s and early 1980s, Earl and Boon Chock carried out a theoretical analysis of the bicyclic cascade system to understand its implications in enzyme regulation. Their work revealed notable advantages of a cyclic cascade system. It amplifies signals and transmits them faster; and it can also sense and respond to continuous changes in the concentration of a number of metabolic intermediates and products. By the time this important theoretical study was finished, Earl had already moved on to another research subject, the regulation of protein degradation.

Video: Boon Chock on the regulation of glutamine synthetase.

Running time: 2:36 minutes 
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E. Garcia and Sue Goo Rhee, 1979

The cyclic cascade system of regulating the activities of glutamine synthetase

 
Boon Chock in his laboratory, circa 1980