Reduced pyridine nucleotides NADH or NADPH pro-vide the reducing power: NH4 -ketoglutarate NADPH H⎯⎯→ glutamate NADP H2O 25.6 This reaction provides an important interface between nit
Trang 1tase, so it no longer binds to nitrogenase The ADP⬊oxidized nitrogenase reductase
complex dissociates, making way for another ATP⬊reduced nitrogenase reductase
complex to bind to nitrogenase Interestingly, nitrogenase reductase is a member of
the G-protein family; G proteins are molecular switches whose operation is driven
by NTP hydrolysis
Nitrogenase is a rather slow enzyme: Its optimal rate of etransfer is about 12 e
pairs per second per enzyme molecule; that is, it reduces only three molecules of
nitrogen gas per second Because its activity is so weak, nitrogen-fixing cells
main-tain large amounts of nitrogenase so that their requirements for reduced N can be
met As much as 5% of the cellular protein may be nitrogenase
The Regulation of Nitrogen Fixation To a first approximation, two regulatory
controls are paramount (Figure 25.8): (1) ADP inhibits the activity of nitrogenase;
thus, as the ATP/ADP ratio drops, nitrogen fixation is blocked (2) NH4 represses
FIGURE 25.7 Ribbon diagram of nitrogenase reductase (the Fe-protein, blue) ⬊nitrogenase (FeMo protein, green) complex The Fe-protein iron-sulfur cluster is shown in yellow, bound ADP in orange The nitrogenase FeMo cofactor is shown in cyan, the P-cluster in red (pdb id 1N2C).
Nitrogenase
Nitrogenase reductase
N
NH2 C O
Nitrogenase reductase
Arg 101 O
ADP
e –
nif gene expression
N2 + + 10 H+ 2 NH4+ + H2
(c)
(b)
(a)
Nicotinamide
Ribose
ADP–ribosyl group
ATP
NAD+
ADP 8
FIGURE 25.8 Regulation of nitrogen fixation (a) ADP inhibits nitrogenase activity (b) NH4 represses nif gene
expression (c) In some organisms, the nitrogenase
complex is regulated by covalent modification ADP– ribosylation of nitrogenase reductase leads to its inactivation.
Trang 2774 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism
the expression of the nif genes, the genes that encode the proteins of the nitrogen-fixing system To date, some 20 nif genes have been identified with the nitrogen fix-ation process Repression of nif gene expression by ammonium, the primary
prod-uct of nitrogen fixation, is an efficient and effective way of shutting down N2fixation when its end product is not needed In addition, in some systems, covalent modifi-cation of nitrogenase reductase leads to its inactivation Inactivation occurs when Arg101of nitrogenase reductase receives an ADP-ribosyl group donated by NAD
25.2 What Is the Metabolic Fate of Ammonium?
Given the prevalence of N atoms in cellular components, it is surprising that only three enzymatic reactions introduce ammonium into organic molecules Of these
three, glutamate dehydrogenase and glutamine synthetase are responsible for most of the ammonium assimilated into carbon compounds The third, carbamoyl-phosphate syn-thetase I, is a mitochondrial enzyme that participates in the urea cycle.
Glutamate dehydrogenase (GDH) catalyzes the reductive amination of
-keto-glutarate to yield glutamate Reduced pyridine nucleotides (NADH or NADPH) pro-vide the reducing power:
NH4 -ketoglutarate NADPH H⎯⎯→
glutamate NADP H2O (25.6) This reaction provides an important interface between nitrogen metabolism and cel-lular pathways of carbon and energy metabolism because -ketoglutarate is a citric acid
cycle intermediate In vertebrates, GDH is an 6-type multimeric enzyme localized in the mitochondrial matrix that uses NADPH as electron donor when operating in the biosynthetic direction (the direction of glutamate synthesis) (Figure 25.9) In contrast, when GDH acts in the catabolic direction to generate -ketoglutarate from glutamate,
NAD, not NADP, is usually the electron acceptor The catabolic activity is allosteri-cally activated by ADP and inhibited by GTP
Glutamine synthetase (GS) catalyzes the ATP-dependent amidation of the
-carboxyl group of glutamate to form glutamine (Figure 25.10) The reaction
pro-ceeds via a -glutamyl-phosphate intermediate, and GS activity depends on the
presence of divalent cations such as Mg2 Glutamine is a major N donor in the
biosynthesis of many organic N compounds such as purines, pyrimidines, and other amino acids, and GS activity is tightly regulated, as we shall soon see The amide-N of glutamine provides the nitrogen atom in these biosyntheses
Carbamoyl-phosphate synthetase I, the third enzyme capable of using ammo-nium to form an N-containing organic compound, catalyzes an early step in the urea cycle Two ATPs are consumed, one in the activation of HCO3 for reaction with ammonium and the other in the phosphorylation of the carbamate formed (see also Figure 25.22):
(25.7)
N-acetylglutamate is an essential allosteric activator for this enzyme.
NH4 HCO3 2 ATP H2N C O PO3 2 ADP Pi 2 H
O
NH3+
C
CH2
CH2
C
+
C
CH2
CH2
C
HC
NH4+
Glu
H2O
-KG
FIGURE 25.9 The glutamate dehydrogenase reaction.
Trang 3The Major Pathways of Ammonium Assimilation Lead
to Glutamine Synthesis
In organisms that enjoy environments rich in nitrogen, GDH and GS acting in
se-quence furnish the principal route of NH4 incorporation (Figure 25.11) However,
GDH has a significantly higher Kmfor NH4 than does GS Consequently, in
organ-isms such as green plants that grow under conditions where little NH4 is available,
GDH is not effective and GS is the only NH4 -assimilative reaction Such a situation
creates the need for an alternative mode of glutamate synthesis to replenish the
glu-tamate consumed by the GS reaction This need is filled by gluglu-tamate synthase (also
known as GOGAT, the acronym for the other name of this enzyme—glutamate⬊
o xo-glutarate amino-transferase) Glutamate synthase catalyzes the reductive
amina-tion of -ketoglutarate using the amide-N of glutamine as the N donor:
Reductant -KG Gln ⎯⎯→ 2 Glu oxidized reductant (25.8)
Two glutamates are formed—one from amination of -ketoglutarate and the other
from deamidation of Gln (Figure 25.12) These glutamates can now serve as
am-monium acceptors for glutamine synthesis by GS Organisms variously use NADH,
NADPH, or reduced ferredoxin as reductant Glutamate synthases are typically
large, complex proteins; in Escherichia coli, GOGAT is an 800-kD flavoprotein
con-taining both FMN and FAD, as well as [4Fe-4S] clusters
NH3+
C
CH2
CH2
C
C
O C
CH2
CH2 C C
+
H
NH4+
NH3+ H
NH2
C
CH2
CH2
C
C
+
NH3+
O
CH2
CH2 C C
C
H
O–
O P O–
+
O–
O P OH
O C
CH2
CH2 C C
H
NH2
ATP
Gln
(a) Glu
Mg2+
(b)
Gln
ADP
(a)
(b)
FIGURE 25.10 (a) The enzymatic reaction catalyzed by
glutamine synthetase (b) The reaction proceeds by (a)
activation of the -carboxyl group of Glu by ATP,
fol-lowed by (b) amidation by NH 4
(a) NH4++-ketoglutarate+ NADPH glutamate GDH + NADP++H2O
(b) Glutamate + NH4++ ATP glutamine GS + ADP +Pi
SUM: 2 NH4++-ketoglutarate+ NADPH + ATP glutamine + NADP++ADP+Pi+H2O
FIGURE 25.11 The GDH/GS pathway of ammo-nium assimilation The sum of these reactions
is the conversion of 1 -ketoglutarate to 1
glu-tamine at the expense of 2 NH 4 , 1 ATP, and
1 NADPH.
Trang 4776 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism
Together, GS and GOGAT constitute a second pathway of ammonium assimilation,
in which GS is the only NH4 -fixing step; the role of GOGAT is to regenerate gluta-mate (Figure 25.13) Note that this pathway consumes 2 equivalents of ATP and
1 NADPH (or similar reductant) per pair of N atoms introduced into Gln, in contrast
to the GDH/GS pathway, in which only 1 ATP and 1 NADPH are consumed per pair
of NH4 fixed Clearly, coping with a nitrogen-limited environment has its cost
25.3 What Regulatory Mechanisms Act on Escherichia coli
Glutamine Synthetase?
As indicated earlier, glutamine plays a pivotal role in nitrogen metabolism by donating its amide nitrogen to the biosynthesis of many important organic N com-pounds Consistent with its metabolic importance, in prokaryotic cells such as
E coli, GS is regulated at three different levels:
1 Its activity is regulated allosterically by feedback inhibition.
2 GS is interconverted between active and inactive forms by covalent modification.
3 Cellular amounts of GS are carefully controlled at the level of gene expression and protein synthesis.
Eukaryotic versions of glutamine synthetase show none of these regulatory features
E coli GS is a 600-kD dodecamer (12-type subunit organization) of identical 52-kD monomers (each monomer contains 468 amino acid residues) These monomers are arranged as a stack of two hexagons (Figure 25.14) The active sites are located at subunit interfaces within the hexagons; these active sites are recognizable in the X-ray crystallographic structure by the pair of divalent
-KG
NH3+
C
CH2
CH2
C
+
C
CH2
CH2 C C
H
Gln
NH3+
O C
CH2
CH2 C C
H
NH2
NADH (yeast, N crassa)+ H+
NADPH (E coli)+ H + or
2 H++ 2 reduced ferredoxin (plants)
+-KG + Gln 2 Glu + NAD
+ NADP + or
2 oxidized ferredoxin
NH3+
C
CH2
CH2 C C
H +
(a) 2 NH4++ 2 ATP + 2 glutamate 2 glutamine GS + 2 ADP +2 Pi
(b) NADPH +-ketoglutarate+ glutamine 2 glutamate GOGAT + NADP+
SUM: 2 NH4++-ketoglutarate+ NADPH + 2 ATP glutamine + NADP++2 ADP+2 Pi
FIGURE 25.12 The glutamate synthase reaction (left), showing the reductants exploited by different organisms
in this reductive amination reaction Structure of glutamate synthase (right) (pdb id 1LM1) FAD is shown in blue, the Fe-S cluster in yellow.
FIGURE 25.13 The GS/GOGAT pathway of
ammonium assimilation The sum of these
reactions results in the conversion of 1
-ketoglutarate to 1 glutamine at the expense
of 2 ATP and 1 NADPH.
Trang 5cations that occupy them Adjacent subunits contribute to each active site, thus
accounting for the fact that GS monomers are catalytically inactive
Glutamine Synthetase Is Allosterically Regulated
Nine distinct feedback inhibitors (Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P, and
glucosamine-6-P) act on GS Gly, Ala, and Ser are key indicators of amino acid
me-tabolism in the cell; each of the other six compounds represents an end product of a
biosynthetic pathway dependent on Gln (Figure 25.15) AMP competes with ATP for
binding at the ATP substrate site Gly, Ala, and Ser compete with Glu for binding at
the active site Carbamoyl-P binds at a site that overlaps both the Glu site and the site
occupied by the -PO4of ATP
Glutamine Synthetase Is Regulated by Covalent Modification
Each GS subunit can be adenylylated at a specific tyrosine residue (Tyr397) in an
ATP-dependent reaction (Figure 25.16) Adenylylation inactivates GS If we
de-fine n as the average number of adenylyl groups per GS molecule, GS activity is
inversely proportional to n The number n varies from 0 (no adenylyl groups) to
12 (every subunit in each GS molecule is adenylylated) Adenylylation of GS is
catalyzed by the converter enzyme ATP ⬊GS⬊adenylyl transferase, or simply adenylyl
transferase (AT) However, whether or not this covalent modification occurs is
de-termined by a highly regulated cycle (Figure 26.17) AT not only catalyzes
adeny-lylation of GS, it also catalyzes deadenyadeny-lylation—the phosphorolytic removal of
the Tyr-linked adenylyl groups as ADP The direction in which AT operates
de-pends on the nature of a regulatory protein, PII, associated with it P IIis a 44-kD
protein (tetramer of 11-kD subunits): The state of PII controls the direction in
FIGURE 25.14 The subunit organization of bacterial
glutamine synthetase (a) Schematic; (b) molecular
structure (note the pairs of metal ions [dark blue] that define the active sites) (pdb id 1FPY).
Trang 6778 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism
P
R
O
O–
+H3N
Mg2+
C H
H C
O
O–
+H3N C
CH3
H C
Glutamine
O
O–
+H3N C
CH2 OH
O
O–
CH2 N N H
N H
O
O–
C
CH2 +NH3
O
NH2
CH2
P
P P
R N N
NH2 N
N
N N
NH2
O–
O P O–
O
O C +H3N
P
+NH3
O
ATP
P
Glutamate +
NH4+
+
Glycine
Alanine
Glutamine synthetase
Serine
Histidine
Tryptophan
Glucosamine-6-P
CTP
Carbamoyl-P
ADP
AMP
FIGURE 25.15 The allosteric regulation of glutamine synthetase activity by feedback inhibition.
CH2
P P
OH
CH2
O
OCH2
N
N N
NH2
ATP
12 + Tyr397in GS
monomer
Glutamine synthetase monomer
Adenylyl transferase
Glutamine synthetase monomer
12
12 Adenylylated Tyr 397 in GS monomer
FIGURE 25.16 Covalent modification of GS: Adenylylation of Tyr 397 in the glutamine synthetase polypeptide via
an ATP-dependent reaction catalyzed by the converter enzyme adenylyl transferase.
Trang 7which AT acts If PII is in its so-called PIIA form, the AT⬊PIIA complex acts to
adenylylate GS When PII is in its so-called PIID form, the AT⬊PIID complex
cat-alyzes the deadenylylation of GS The active sites of AT⬊PIIAand AT⬊PIIDare
dif-ferent, consistent with the difference in their catalytic roles In addition, the
AT⬊PIIA and AT⬊PIID complexes are allosterically regulated in a reciprocal
fash-ion by the effectors -KG and Gln Gln activates AT⬊PIIA activity and inhibits
AT⬊PIIDactivity; the effect of -KG on the activities of these two complexes is
di-ametrically opposite (Figure 25.17) Further, Gln favors conversion of PIIDto PIIA,
whereas-ketoglutarate favors the PIIDover the PIIAform
Clearly, the determining factor regarding the degree of adenylylation, n, and
hence the relative activity of GS, is the [Gln]/[-KG] ratio A high [Gln] level
sig-nals cellular nitrogen sufficiency, and GS becomes adenylylated and inactivated In
contrast, a high [-KG] level is an indication of nitrogen limitation and a need for
ammonium fixation by GS
Glutamine Synthetase Is Regulated Through Gene Expression
The gene that encodes the GS subunit in E coli is designated GlnA The GlnA gene
is actively transcribed to yield GS mRNA for translation and synthesis of GS protein
only if a specific transcriptional enhancer, NRI ,is in its phosphorylated form, NRI-P In
turn, NRIis phosphorylated in an ATP-dependent reaction catalyzed by NR II ,a
pro-tein kinase (Figure 25.18) However, if NRIIis complexed with PIIA, it acts not as a
kinase but as a phosphatase, and the transcriptionally active form of NRI, namely
NRI-P, is converted back to NRIwith the result that GlnA transcription halts Recall
from the foregoing discussion that a high [Gln]/[-KG] ratio favors PIIAat the
ex-pense of PIID Under such conditions, GS gene expression is not necessary
25.4 How Do Organisms Synthesize Amino Acids?
Organisms show substantial differences in their capacity to synthesize the
20 amino acids common to proteins Typically, plants and microorganisms can
form all of their nitrogenous metabolites, including all of the amino acids, from
P P
P
+
+
ATP
12
Adenylyl transferase:
PIIA complex
Adenylyl transferase:
PIID complex
12 ADP
Less active GS:
Tyr397–O– AMP adenylylated
-KG Gln
-KG Gln
Active GS:
Tyr397 unadenylylated
Highly sensitive
to feedback inhibition
*State of PII controls adenylyl transferase direction
FIGURE 25.17 The cyclic cascade system regulating the covalent modification of GS.
+
NRII
H2O
P
NRII:PIIA
P
activates GlnA
transcription; GS
is synthesized
FIGURE 25.18 Transcriptional regulation of GlnA
expres-sion through the reversible phosphorylation of NR I
Trang 8780 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism
inorganic forms of N such as NH4 and NO3 In these organisms, the -amino
group for all amino acids is derived from glutamate, usually via transamination of the corresponding -keto acid analog of the amino acid (Figure 25.19) In many
cases, amino acid biosynthesis is thus a matter of synthesizing the appropriate
-keto acid carbon skeleton, followed by transamination with Glu The amino
acids can be classified according to the source of intermediates for the -keto acid
biosynthesis (Table 25.1) For example, the amino acids Glu, Gln, Pro, and Arg
CH2
C COO–
NH3+ COO–
COO–
COO–
COO–
COO–
COO–
COO–
COO–
CH2
CH2 C H
+
R O
CH2
CH2
C COO– +
R
NH3+
CH2
CH2 C H
+ C COO–
O
CH2
CH2
+ C COO–
CH2
Glutamate -Keto acid -KG -Amino acid
Glutamate Oxaloacetate -KG Aspartate
Pyridoxal phosphate-dependent aminotransferase
Glutamate-aspartate aminotransferase
ACTIVE FIGURE 25.19
Glutamate-dependent transamination of -keto acid carbon
skele-tons is a primary mechanism for amino acid synthesis.
The transamination of oxaloacetate by glutamate to
yield aspartate and -ketoglutarate is a prime example.
Test yourself on the concepts in this figure at
www.cengage.com/login.
Lysine*
Phosphoenolpyruvate and Erythrose-4-P Family The aromatic amino acids
Phenylalanine Tyrosine Tryptophan
The remaining amino acid, histidine, is derived from PRPP
(5-phosphoribosyl-1-pyrophosphate) and ATP
TABLE 25.1 The Grouping of Amino Acids into Families According to the Metabolic Intermediates
That Serve as Their Progenitors
Trang 9(and, in some instances, Lys) are all members of the -ketoglutarate family
be-cause they are all derived from the citric acid cycle intermediate -ketoglutarate.
We return to this classification scheme later when we discuss the individual
biosynthetic pathways
Amino Acids Are Formed from ␣-Keto Acids by Transamination
Transamination involves transfer of an -amino group from an amino acid to the
-keto position of an -keto acid (Figure 25.19) In the process, the amino donor
be-comes an -keto acid while the -keto acid acceptor becomes an -amino acid:
Amino acid -keto acid ⎯⎯→ -keto acid amino acid (25.9)
HUMAN BIOCHEMISTRY
Human Dietary Requirements for Amino Acids
Humans can synthesize only 10 of the 20 common amino acids
(see table below); the others must be obtained in the diet Those
that can be synthesized are classified as nonessential, meaning it
is not essential that these amino acids be part of the diet In
ef-fect, humans can synthesize the -keto acid analogs of
nonessen-tial amino acids and form the amino acids by transamination In
contrast, humans are incapable of constructing the carbon
skele-tons of essential amino acids, so they must rely on dietary sources
for these essential metabolites Excess dietary amino acids cannot
be stored for future use, nor are they excreted unused Instead,
they are converted to common metabolic intermediates that can
be either oxidized by the citric acid cycle to generate metabolic
energy or used to form glucose (see Section 25.5)
Since autotrophic cells (and many prokaryotic cells) synthesize
all 20 amino acids, several questions arise regarding human
di-etary requirements for amino acids First, why is it that humans
lack the ability to do what other organisms can do? The answer is
that, over evolutionary time, human diets provided adequate
amounts of those amino acids classified now as “essential.” Thus,
the loss of the metabolic pathways for synthesis of “essential”
amino acids did not impair the fitness of humans That is,
synthe-sizing “essential” amino acids became superfluous, and no
evolu-tionary pressure operated on humans to retain the genes for these
pathways A second question now emerges: Are there significant
differences between synthesis of amino acids human can make
(the so-called “nonessential” amino acids) and those they can’t?
The big table summarizes amino acid biosynthesis from citric acid cycle intermediates in terms of the number of reactions needed to make an amino acid from a TCA cycle intermediate.* Nonessen-tial amino acids are shown in blue; essenNonessen-tial amino acids in red Two conclusions stand out: Nonessential amino acids require fewer reaction steps for synthesis than essential amino acids, and nonessential amino acids tend to be more abundantly represented
in proteins than essential amino acids Thus, evolutionary loss was not random: The biosynthetic pathways lost were those for amino acids requiring the most reaction steps
*From Srinivasan, V., Morowitz, H., and Smith, E., 2007 Essential amino
acids, from LUCA to LUCY Complexity 13:8–9.
Histidine* Asparagine
Phenylalanine Glycine
*Arginine and histidine are essential in the diets of juveniles, not adults.
†Tyrosine is classified as nonessential only because it is readily formed from essential
phenylalanine.
Essential and Nonessential Amino Acids in Humans
Amino Acid Reaction Steps Mole % in Proteins†
†Mole percentages are taken from amino acid representations among proteins in the Swiss-Prot protein knowledgebase: ca.expasy.org/sprot.
‡Note that “nonessential” tyrosine can only be made from “essential” phenylanine.
Nonessential Amino Acids Require Fewer Reactions for Synthesis
Trang 10782 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism
The predominant amino acid/-keto acid pair in these reactions is glutamate/
-ketoglutarate, with the net effect that glutamate is the primary amino donor
for the synthesis of amino acids Transamination reactions are catalyzed by
aminotransferases (the preferred name for enzymes formerly termed transami-nases) Aminotransferases are named according to their amino acid substrates, as in
glutamate–aspartate aminotransferase. Aminotransferases are prime examples of enzymes that catalyze double displacement (ping-pong)–type bisubstrate reactions (see Figure 13.23)
The Pathways of Amino Acid Biosynthesis Can Be Organized into Families
As indicated in Table 25.1, the amino acids can be grouped into families on the basis of the metabolic intermediates that serve as their precursors
A DEEPER LOOK
The Mechanism of the Aminotransferase (Transamination) Reaction
The aminotransferase (transamination) reaction is a workhorse in
biological systems It provides a general means for exchange of
nitrogen between amino acids and -keto acids This vital reaction
is catalyzed by pyridoxal phosphate (PLP) The mechanism
in-volves loss of the C proton, followed by an aldimine–ketimine
tautomerization—literally a “flip-flop” of the Schiff base double
bond from the pyridoxal aldehyde carbon to the -carbon of the
amino acid substrate This is followed by hydrolysis of the ketimine intermediate to yield the product -keto acid Left in the active
site is a pyridoxamine phosphate intermediate, which combines with another (substrate) -keto acid to form a second ketimine,
which rearranges to form an aldamine, followed by release as an amino acid Transaldiminization with a lysine at the active site completes the reaction
+
H
NH3+
N HC +
CH3 N
H +
2 –O3PO
N
O
+ –
N H +
2 –O3PO
O
NH2
H2C
O–
CH3 N
H +
2 –O3PO
O
N
H2C +
CH3 N
H +
2 –O3PO +
H
NH3+
H
N HC +
CH3 N
H +
2 –O3PO
H
CH3
H
O–
H +
H +
H2O
H2O
C
2– O3PO
OH
CH3 N
H +
Lysine
H
O–
H
O–
Ketimine Aldimine
Transamination intermediate
Pyridoxamine
E•PLP complex
E-PLP complex
䊱 The mechanism of PLP-catalyzed transamination reactions.