In 1960, Earl went on sabbatical leave
to Europe, and this turned out to be a fruitful research
experience. Working in Feodor
Lynen's laboratory in Munich for half a year,
Earl discovered a biochemical reaction dependent upon
the vitamin B12coenzyme.
Subsequently, at the Pasteur Institute in Paris, he
collaborated with Georges Cohen and others on investigating
the regulation of activities of
enzyme that catalyzes the conversion of
aspartate, an amino
acid, to its phosphate derivative. At that time it was
well known that this conversion was the first common
step in a "branched pathway" that led to the biosynthesis
of three different amino acids-lysine, threonine, and
(A typical example of the branched pathway
where A is a precursor for the biosynthesis of three
different products, X, Y, and Z. These products may
inhibit individually the first common step of A to B).
Earl and his collaborators separated
two different kinds of aspartokinase from E. coli
extracts and obtained evidence suggesting the existence
of still another. They further demonstrated that each
one of these multiple enzymes can be regulated individually
by a particular product of one of the branches in the
Since amino acids are the building blocks
of protein, they are readily obtained in the process
of digesting or degrading protein supplied by foods.
But the organism is also capable of synthesizing amino
acids from other molecules. For example, bacteria such
as E. coli can make the entire basic set of
amino acids. Humans can make some of them, which are
called " non-essential amino acids," whereas
the others that must be supplied in the diet are termed
" essential amino acids." When and how many
amino acids are to be produced is mainly dependent upon
the activity of their biosynthetic enzymes, and the
enzymatic activity is often controlled by the final
products of the pathway, as seen in the biosynthesis
of lysine, threonine, and methionine.
After returning from Europe, Earl resumed
his previous research on the metabolism of heterocyclic
compounds. This investigation was interrupted in 1963,
however, when a postdoctoral fellow, Clifford Woolfolk,
joined his laboratory. Although Woolfolk came with a
strong desire to follow up Earl's work in Paris on the
regulation of aspartokinase, he was advised by Earl
to search for another enzyme that could catalyze the
first common step in a branched pathway. In 1964, Earl
was delighted to find that, among the three candidates
Woolfolk suggested, glutamine synthetase was involved
in a branched pathway, its activity being regulated
by several end products. As Earl later recollected,
this "exciting discovery initiated a dramatic change
in the focus of much of the research in the Laboratory
of Biochemistry and for almost 35 years has occupied
the time and energies of numerous highly talented postdoctoral
fellows, visiting scientists, and senior associates."
It was the beginning of a highly successful, productive
research program, in which Earl demonstrated his ingenuity
not only as a scientist but also as a leader of many
Glutamine synthetase. Because
of its fine control system, this enzyme
is nicknamed the "virtual molecular computer"
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 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."
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)
toand also its detachment fromspecific 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.