Chem*3560
Lecture 21:
Fatty acid synthase
Fatty acids are made by a multienzyme complex called fatty acid synthase, which contains seven
distinct catalytic centres. Fatty acid synthase is arranged around a central acyl carrier protein,
which contains a protein bound pantetheine chain similar to the long chain of Coenzyme A.
The pantetheine chain is bonded to a serine side chain
of acyl carrier protein (ACP). Pantetheine acts as a long
"arm" that fixes the growing acyl chain in the fatty acid
synthase, but gives it sufficient mobility in the enzyme to
visit the different catalytic sites (Lehninger p.772-776).
The catalytic cycle starts with ketoacylACP synthase
(KS) which condenses malonyl ACP to the growing acyl
chain (R is a simple saturated hydrocarbon chain). The
condensation displaces the extra carboxylate from the
malonyl-ACP, so that decarboxylation provides driving
force for the reaction (Lehninger Fig. 21-2).
Next, the ketone group is reduced to a hydroxyl group
by ketoacyl ACP reductase (KR), which requires
NADPH as H donor.
H2 O is eliminated by hydroxyacyl ACP dehydratase
(HD) to give the enoyl-ACP.
A second reduction step also uses NADPH and is
catalyzed by enoyl ACP reductase (ER), yielding a
saturated acyl chain that is two C atoms longer than the
original R.
These four reactions effectively reverse the β-oxidation process. Fatty acid synthase however requires
two additional reactions in order to keep the growing chain on the central ACP.
The catalytic site of ketoacyl ACP synthase includes a Cys side chain which contributes an auxilary
-SH site for the saturated acyl chain. This is indicated in the reaction scheme as R-CO-S-Enz or
HS-Enz.
The enzyme acyl transferase (AT) plays
a dual role:
If ACP is currently unoccupied, acyl
transferase accepts acetyl CoA as a
substrate, and transfers the acetyl group
to the HS-Enz position (left). This acts to
start a new chain.
If ACP is occupied by saturated
acyl-S-ACP (the product of enoyl ACP
reductase), acyl transferase transfers the
saturated acyl from ACP to the auxilary
HS-Enz site (right). This allows for
another cycle of elongation of an
existing chain, and makes HS-ACP
available for the next reaction.
The enzyme malonyl CoA-ACP transferase (MT) accepts malonyl CoA as substrate, and transfers
the malonyl group to HS-ACP to prepare for the next ketoacyl ACP synthase reaction.
The ketoacyl ACP synthase then transfers the acyl group from acyl-S-Enz onto the malonyl CH2 , and
the resulting ketoacyl product remains bonded to ACP. This means that the growing acyl chain can go
through repeated elongation cycles without ever leaving the fatty acid synthase.
Fatty acid synthase repeats the
reaction cycle seven times
The first cycle starts with acetyl CoA (2
C atoms), which is converted to
acetyl-S-Enz in the KS catalytic site, and
then elongated by reaction with
malonyl-S-ACP.
Since the malonyl loses its extra
carboxylate group, this reaction adds 2
more C atoms to the growing chain. The
elongation cycle repeats for a total of 7
cycles, to give the C16 product
palmitoyl-S-ACP.
Chain length is monitored by the seventh
catalytic site in fatty acid synthase,
thioesterase (TE).
When the acyl chain length reaches 16 C
atoms, thioesterase hydrolyses the
acyl-S-ACP bond to release free
palmitate, C15 H31 CO2 – .
palmitoyl-S-ACP + H2 O → palmitate + H+ + HS-ACP
Organization of fatty acid synthase
Fatty acid synthase consists of the seven catalytic centres arranged
around a central ACP. In bacteria such as E.coli, ACP is a small
protein of mass 8.8 kDa. Six additional catalytic subunits are
arranged around the central ACP (Lehninger p. 777 and Figs. 21-5
to 21-7; however these figures should be regarded as schematic
rather than realistic).
The pantetheine arm is anchored into ACP, but is long enough to
allow covalently bound substrate and intermediates reach all seven
catalytic sites. After the acetyl group is first attached to the KS HS-Enz site, the intermediates remain
covalently bonded to the enzyme unitl palmitate is finally released. This confines the intermediates to
remain within close range of their target catalytic sites and eliminates the inefficiency of random diffusion
of intermediates into and out of the surrounding solution.
Mammalian fatty acid synthase is a dimer of two
polypeptides of 240 kDa each. Each polypeptide
contains eight domains that represent the seven catalytic
centres plus an integral ACP domain.
The pantetheine arm allows intermediates to reach KR,
ER and HD sites on the same polypeptide, but KS, AT
and MT sites on the opposite polypeptide are closer
than those on the same polypeptide.
Overall cost of fatty acid synthesis
The overall fatty acid synthase equation for seven cycles
acetyl-CoA + 7 malonyl-CoA + 14 NADPH → palmitate + 7 CO2 + 8 HSCoA + 14 NADP+
+ 14 H+
+ 6 H2 O
Overall cost of synthesis in bacteria includes 7 ATP as the energy cost of making 7 malonyl-CoA:
acetyl CoA carboxylase
7 acetyl-CoA + 7 ATP + 7 CO2 + 7 H2 O → 7 malonyl-CoA + 7 H+ + 7 ADP + 7 Pi
Overall cost of synthesis in mammalian cells includes an additional 8 ATP required for citrate lyase
citrate lyase
8 citrate + 8 ATP + 8 HSCoA → 8 acetyl-CoA + 8 oxaloacetate + 8 ADP + 8 Pi
Sources of NADPH
There are three common sources of NADPH (other than in higher plants, where NADPH is produced
directly by photosynthesis). The pentose phosphate cycle is a major source of NADPH.
Depending on organism and tissue, two other common sources of NADPH may be used:
malate
2–
malate dehydrogenase (decarboxylating) or malic enzyme
+ NADP+ → pyruvate – + CO2 + NADPH
The enzyme makes oxaloacetate as a bound intermediate, and then decarboxylates it to release
pyruvate as a product. The decarboxylation provides the driving force needed to produce excess
NADPH.
Enz:[oxaloacetate] → pyruvate + CO2
Many organisms also contain an NADP + dependent isozyme of isocitrate dehydrogenase.
isocitrate dehydrogenase (NADP + dependent)
isocitrate + NADP+ → α -ketoglutarate + CO2 + NADPH
Unlike the NAD+ dependent isozyme, this isozyme does not require ADP for activation, so stays active
when low levels of ADP cause the normal TCA cycle isocitrate dehydrogenase to lose activity.
isocitrate dehydrogenase
isocitrate + NAD + –\\→
→ α -ketoglutarate + CO2 + NADH
no activity at low [ADP]