Amino Acid Biosynthesis Is Regulated by Feedback Inhibition

Figure 24.19. Structure of Tryptophan Synthetase. The structure of the complex formed by one α subunit and one β
subunit. PLP is bound to the β subunit.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.2. Amino Acids Are Made from Intermediates of the Citric Acid Cycle and Other Major Pathways
Figure 24.20. Substrate Channeling. A 25-Å tunnel runs from the active site of the α subunit of tryptophan synthetase
(yellow) to the PLP cofactor (red) in the active site of the β subunit (blue).
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
The rate of synthesis of amino acids depends mainly on the amounts of the biosynthetic enzymes and on their activities.
We now consider the control of enzymatic activity. The regulation of enzyme synthesis will be discussed in Chapter 31.
In a biosynthetic pathway, the first irreversible reaction, called the committed step, is usually an important regulatory
site. The final product of the pathway (Z) often inhibits the enzyme that catalyzes the committed step (A
B).
This kind of control is essential for the conservation of building blocks and metabolic energy. Consider the biosynthesis
of serine (Section 24.2.5). The committed step in this pathway is the oxidation of 3-phosphoglycerate, catalyzed by the
enzyme 3-phosphoglycerate dehydrogenase. The E. coli enzyme is a tetramer of four identical subunits, each comprising
a catalytic domain and a serine-binding regulatory domain (Figure 24.21). The regulatory domains of two subunits
interact to form a dimeric serine-binding regulatory unit so that the tetrameric enzyme contains two such regulatory
units. Each unit is capable of binding two serine molecules. The binding of serine to a regulatory site reduces the value
of V max for the enzyme; an enzyme bound to four molecules of serine is essentially inactive. Thus, if serine is abundant
in the cell, the enzyme activity is inhibited, and so 3-phosphoglycerate, a key building block that can be used for other
processes, is not wasted.
24.3.1. Branched Pathways Require Sophisticated Regulation
The regulation of branched pathways is more complicated because the concentration of two products must be accounted
for. In fact, several intricate feedback mechanisms have been found in branched biosynthetic pathways.
Feedback Inhibition and Activation.
Consider, for example, the biosynthesis of the amino acids valine, leucine, and isoleucine. A common intermediate,
hydroxyethyl thiamine pyrophosphate (hydroxyethyl-TPP; Section 17.1.1), initiates the pathways leading to all three of
these amino acids. Hydroxyethyl-TPP can react with α-ketobutyrate in the initial step for the synthesis of isoleucine.
Alternatively, hydroxyethyl-TPP can react with pyruvate in the committed step for the pathways leading to valine and
leucine. Thus, the relative concentrations of α-ketobutyrate and pyruvate determine how much isoleucine is produced
compared with valine and leucine. Threonine deaminase, the PLP enzyme that catalyzes the formation of α-ketobutyrate,
is allosterically inhibited by isoleucine (Figure 24.22). This enzyme is also allosterically activated by valine. Thus, this
enzyme is inhibited by the product of the pathway that it initiates and is activated by the end product of a competitive
pathway. This mechanism balances the amounts of different amino acids that are synthesized.
The regulatory domain in threonine deaminase is very similar in structure to the dimeric regulatory domain in 3phosphoglycerate dehydrogenase (Figure 24.23). In this case, the two half regulatory domains are fused into a single unit
with two differentiated amino acid-binding sites, one for isoleucine and the other for valine. Sequence analysis shows
that similar regulatory domains are present in other amino acid biosynthetic enzymes. The similarities suggest that
feedback-inhibition processes may have evolved by the linkage of specific regulatory domains to the catalytic domains of
biosynthetic enzymes.
Enzyme Multiplicity.
Sophisticated regulation can also evolve by duplication of the genes encoding the biosynthetic enzymes. For example,
the phosphorylation of aspartate is the committed step in the biosynthesis of threonine, methionine, and lysine. Three
distinct aspartokinases catalyze this reaction in E. coli, an example of a regulatory mechanism called enzyme multiplicity.
(Figure 24.24). The catalytic domains of these enzymes show approximately 30% sequence identity. Although the
mechanisms of catalysis are essentially identical, their activities are regulated differently: one enzyme is not subject to
feedback inhibition, another is inhibited by threonine, and the third is inhibited by lysine.
Cumulative Feedback Inhibition
The regulation of glutamine synthetase in E. coli is a striking example of cumulative feedback inhibition. Recall that
glutamine is synthesized from glutamate, NH4 +, and ATP. Glutamine synthetase consists of 12 identical 50-kd subunits
arranged in two hexagonal rings that face each other (Figure 24.25). Earl Stadtman showed that this enzyme regulates
the flow of nitrogen and hence plays a key role in controlling bacterial metabolism. The amide group of glutamine is a
source of nitrogen in the biosyntheses of a variety of compounds, such as tryptophan, histidine, carbamoyl phosphate,
glucosamine 6-phosphate, cytidine triphosphate, and adenosine monophosphate. Glutamine synthetase is cumulatively
inhibited by each of these final products of glutamine metabolism, as well as by alanine and glycine. In cumulative
inhibition, each inhibitor can reduce the activity of the enzyme, even when other inhibitors are bound at saturating
levels. The enzymatic activity of glutamine synthetase is switched off almost completely when all final products are
bound to the enzyme.
24.3.2. The Activity of Glutamine Synthetase Is Modulated by an Enzymatic Cascade
The activity of glutamine synthetase is also controlled by reversible covalent modification the attachment of an AMP
unit by a phosphodiester bond to the hydroxyl group of a specific tyrosine residue in each subunit (Figure 24.26). This
adenylylated enzyme is less active and more susceptible to cumulative feedback inhibition than is the deadenylylated
form. The covalently attached AMP unit is removed from the adenylylated enzyme by phosphorolysis. The attachment of
an AMP unit is the final step in an enzymatic cascade that is initiated several steps back by reactants and immediate
products in glutamine synthesis.
The adenylation and phosphorolysis reactions are catalyzed by the same enzyme, adenylyl transferase. Sequence
analysis indicates that this adenylyl transferase comprises two homologous halves, suggesting that one half catalyzes the
adenylation reaction and the other half the phospholytic de-adenylation reaction. What determines whether an AMP unit
is added or removed? The specificity of adenylyl transferase is controlled by a regulatory protein (designated P or PII), a
trimeric protein that can exist in two forms, PA and PD (Figure 24.27). The complex of PA and adenylyl transferase
catalyzes the attachment of an AMP unit to glutamine synthetase, which reduces its activity. Conversely, the complex of
PD and adenylyl transferase removes AMP from the adenylylated enzyme.
This brings us to another level of reversible covalent modification. PA is converted into PD by the attachment of uridine
monophosphate to a specific tyrosine residue (Figure 24.28). This reaction, which is catalyzed by uridylyl transferase, is
stimulated by ATP and α-ketoglutarate, whereas it is inhibited by glutamine. In turn, the UMP units on PD are removed
by hydrolysis, a reaction promoted by glutamine and inhibited by α-ketoglutarate. These opposing catalytic activities are
present on a single polypeptide chain, homologous to adenylyl transferase, and are controlled so that the enzyme does
not simultaneously catalyze uridylylation and hydrolysis.
Why is an enzymatic cascade used to regulate glutamine synthetase? One advantage of a cascade is that it amplifies
signals, as in blood clotting and the control of glycogen metabolism (Sections 10.5.5 and 21.3.1). Another advantage is
that the potential for allosteric control is markedly increased when each enzyme in the cascade is an independent target
for regulation. The integration of nitrogen metabolism in a cell requires that a large number of input signals be detected
and processed. In addition, the regulatory protein P also participates in regulating the transcription of genes for glutamine
synthetase and other enzymes taking part in nitrogen metabolism. The evolution of a cascade provided many more
regulatory sites and made possible a finer tuning of the flow of nitrogen in the cell.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
Figure 24.21. Structure of 3-Phosphoglycerate Dehydrogenase. This enzyme, which catalyzes the committed step in
the serine biosynthetic pathway, includes a serine-binding regulatory domain. Serine binding to this domain
reduces the activity of the enzyme.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
Figure 24.22. Regulation of Threonine Deaminase. Threonine is converted into α-ketobutyrate in the committed step
leading to the synthesis of isoleucine. The enzyme that catalyzes this step, threonine deaminase, is inhibited by
isoleucine and activated by valine, the product of a parallel pathway.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
Figure 24.23. A Recurring Regulatory Domain. The regulatory domain formed by two subunits of 3-phosphoglycerate
dehydrogenase is structurally related to the single-chain regulatory domain of threonine deaminase. Sequence
analyses have revealed this amino acid-binding regulatory domain to be present in other enzymes as well.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
Figure 24.24. Domain Structures of Three Aspartokinases. Each catalyzes the committed step in the biosynthesis of a
different amino acid: (top) methionine, (middle) threonine, and (bottom) lysine. They have a catalytic domain in
common but differ in their regulatory domains.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
Figure 24.25. Structure of Glutamine Synthetase. Glutamine synthetase consists of 12 identical subunits arranged in
two rings of six subunits. The active sites are indicated by the presence of manganese ions (two yellow spheres).
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
24.3. Amino Acid Biosynthesis Is Regulated by Feedback Inhibition
Figure 24.26. Regulation by Adenylation. (A) A specific tyrosine residue in each subunit in glutamine synthetase is
modified by adenylation. (B) Adenylation of tyrosine is catalyzed by a complex of adenylyl transferase (AT) and one
form of a regulatory protein (PA). The same enzyme catalyzes deadenylation when it is complexed with the other form
(PD) of the regulatory protein.