Page 30 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

P1: ZCK Final Pages
Encyclopedia of Physical Science and Technology EN005G-231 June 15, 2001 20:46

Enzyme Mechanisms 637

In the transamination reaction, formation of the aldi- At the core of cytochrome P450 is an iron porphyrin, or
mine intermediate between aspartate and PLP and its heme, group, protoporphyrin IX, which is depicted below.
deprotonation proceeds as described above for the race-
mases. However, reprotonation occurs not at the same COO
carbon as in the racemization mechanism but at a posi-
tion adjacent to the PLP heterocycle.
H H CO 2 N
O 2 C CO 2 O 2 C CO 2 O 2 C COO
H N H N O NH 2
H H N Fe N
H
2 O 3 PO 2 O 3 PO H 2 O 2 O 3 PO
O H H O O
N
N N N
H H H
Hydrolysis releases the product oxaloacetate and gener-
ates a new form of the cofactor called pyridoxamine. The
reverse reaction is then carried out on the other substrate,
α-ketoglutarate, forming glutamate and regenerating the In the structure in Fig. 9, the iron porphyrin is shown in
PLP cofactor. black.
Reactions at the β-position (for example, in threonine Cytochrome P450 is a redox catalyst. The multiple
dehydatase) or the γ -position (in methionine-γ -lyase) available oxidation states allow the cofactor to accept and
also proceed by means of formation of an aldimine inter- donate electrons during different stages of the catalytic
mediate with the α-carbon of an α-amino acid. Such a sur- cycle. Since the early 1970s, this enzyme and its relatives
vey of PLP-dependent enzymes illustrates the important have been the subject of intense study by, among oth-
point that one cofactor can be used for different kinds of ers, enzymologists, toxicologists, biophysical chemists,
transformations. The reactions described all go through a and inorganic chemists. The latter have tried to model
common aldimine intermediate, with the ultimate course the chemistry of cytochrome P450 with synthetic small
of the reaction being controlled by the appropriate sub- molecules with the twin goals of mimicking its activity
stratespecificityandpositioningofaminoacidsidechains. and understanding how the enzyme itself works. Work-
This flexibility allows nature to expand its chemical reper- ing in parallel, biophysical chemists and enzymologists
toire with a relatively small set of cofactors. have performed many steady-state and pretransient ki-
There are other organic cofactors such as thiamine netic studies such as the ones already discussed, which
pyrophosphate and biotin that participate in carbon– have contributed to a working model for the mechanism
carbon bond formation and cleavage, cofactors that shown in Fig. 10.
participate in reduction/oxidation, or redox, reactions Although this mechanism is in some senses more
such as nicotinamide and flavin moieties discussed in complicated than those that we have discussed, the same
some of the earlier examples, and still others that are concepts apply. Starting at the top of the cycle, in the
metal based such as vitamin B 12 and porphyrin, which is resting state of the enzyme the iron is in the +3 oxidation
our next topic. state and is bound by water. Substrate docks to its specific
binding site and displaces water to start the catalytic
F. Cytochrome P450 cycle, and an electron is then introduced to reduce the
iron to the +2 oxidation state. The dashed line is meant
Adifferent kind of cofactor from PLP is responsible for the
to indicate association of the substrate with the active
chemistry of cytochrome P450 (Fig. 9), an enzyme which
site, not an actual bond to the iron. The requirement that
oxidizes hydrocarbons. It is known as a mixed-function
substrate bind before reduction occurs is a control feature
oxidase, or monooxygenase, because one oxygen atom
which prevents formation of very active and potentially
from molecular oxygen is incorporated into the product
damaging species in the absence of substrate. Oxygen
while the other goes on to form water. Cytochrome P450 in
then binds and accepts an electron from the iron, and
the liver, for example, oxidizes and detoxifies many kinds introduction of another electron and two protons allows
of substances that would otherwise be poisonous. One one atom of dioxygen to be released as water, which
such well-studied reaction, the hydroxylation of camphor, leaves behind a very active high valent (formally iron
is depicted below.
5+) species. What follows is known as a radical rebound
step. A hydrogen atom is removed from the substrate and
transferred to the terminal oxygen atom, which produces

O 2 , 2H , 2e
O O H 2 O a substrate radical. The radical recombines with the new
OH hydroxo moiety to form the hydroxylated product, which