474
H. Hirao
4.1 Studies of Heme Enzymes
For example, P450s use the short-lived Cpd I species for oxidation reactions, and the
nature of Cpd I and its reactivity in metabolic reactions have been widely explored
theoretically [16–18]. Other than metabolism, mechanism-based inactivation (MBI)
of P450s is a problem of practical importance that presents a serious concern in
virtually all drug discovery projects. Using truncated small models and DFT calculations, we have investigated the mechanisms of several MBI processes [61–65].
These calculations provided valuable insights into how Cpd I reacts with inhibitor
molecules to cause P450 inhibition.
P450s also sometimes play essential roles in synthesizing signaling molecules.
For example, CYP19A1 (aromatase) is known to catalyze the androgen-to-estrogen
conversion in three major reaction steps. Although the first and second steps are
already well understood, there has been controversy over whether the active species
in the third step is Cpd I or ferric peroxide (Fig. 11a, b). Until recently, many people
had believed that ferric peroxide should be the active species because experiments
supported it. In this ferric peroxide mechanism, one of the two oxygen atoms from
O 2 is incorporated into the formic acid product (Fig. 11c). However, recent
18 Olabeling experiments could not find the O 2 -derived oxygen in formic acid [66], which
indicated that the Cpd I mechanism rather than the ferric peroxide mechanism is
operative. We performed QM/MM calculations to answer the question of how Cpd
I could affect the third step of the aromatase reaction [67]. The QM/MM enzyme
model was built from a crystal structure of human CYP19A1 (PDB code 3EQM)
[68]. Based on the QM/MM results, we proposed a novel mechanism in which Cpd I
abstracts a hydrogen atom from the O–H bond of the gem-diol form of the substrate
to yield the aromatized product. The QM/MM calculations also suggested that Cpd
I can abstract the hydrogen atom of the C–H bond of gem-diol or aldehyde to form
a carboxylic acid product, although the fate of the O 2 -derived oxygen was different
in these reactions, which explained how and why the carboxylic acid was obtained
in the experimental condition.
In our studies of P450s, we are also interested in how different groups within
enzymes interact with each other. Thus, we have tried to identify the origins of
interactions with the aid of various methods [69–71]. Interaction energies between
fragments were decomposed into electrostatic and several other terms.
4.2 Studies of Nonheme Enzymes
We have been applying the DFT and QM/MM methods to many other metalloenzymes [72–77]. Most recently, we have studied the mechanism of CO oxidation
catalyzed by Mo–Cu carbon monoxide dehydrogenase (Mo–Cu CODH) using the
ONIOM QM/MM method [78]. The QM/MM enzyme model was built from a crystal
structure (PDB code 1N5W) [79]. Over the past two decades, the mechanism of the
H. Hirao
4.1 Studies of Heme Enzymes
For example, P450s use the short-lived Cpd I species for oxidation reactions, and the
nature of Cpd I and its reactivity in metabolic reactions have been widely explored
theoretically [16–18]. Other than metabolism, mechanism-based inactivation (MBI)
of P450s is a problem of practical importance that presents a serious concern in
virtually all drug discovery projects. Using truncated small models and DFT calculations, we have investigated the mechanisms of several MBI processes [61–65].
These calculations provided valuable insights into how Cpd I reacts with inhibitor
molecules to cause P450 inhibition.
P450s also sometimes play essential roles in synthesizing signaling molecules.
For example, CYP19A1 (aromatase) is known to catalyze the androgen-to-estrogen
conversion in three major reaction steps. Although the first and second steps are
already well understood, there has been controversy over whether the active species
in the third step is Cpd I or ferric peroxide (Fig. 11a, b). Until recently, many people
had believed that ferric peroxide should be the active species because experiments
supported it. In this ferric peroxide mechanism, one of the two oxygen atoms from
O 2 is incorporated into the formic acid product (Fig. 11c). However, recent
18 Olabeling experiments could not find the O 2 -derived oxygen in formic acid [66], which
indicated that the Cpd I mechanism rather than the ferric peroxide mechanism is
operative. We performed QM/MM calculations to answer the question of how Cpd
I could affect the third step of the aromatase reaction [67]. The QM/MM enzyme
model was built from a crystal structure of human CYP19A1 (PDB code 3EQM)
[68]. Based on the QM/MM results, we proposed a novel mechanism in which Cpd I
abstracts a hydrogen atom from the O–H bond of the gem-diol form of the substrate
to yield the aromatized product. The QM/MM calculations also suggested that Cpd
I can abstract the hydrogen atom of the C–H bond of gem-diol or aldehyde to form
a carboxylic acid product, although the fate of the O 2 -derived oxygen was different
in these reactions, which explained how and why the carboxylic acid was obtained
in the experimental condition.
In our studies of P450s, we are also interested in how different groups within
enzymes interact with each other. Thus, we have tried to identify the origins of
interactions with the aid of various methods [69–71]. Interaction energies between
fragments were decomposed into electrostatic and several other terms.
4.2 Studies of Nonheme Enzymes
We have been applying the DFT and QM/MM methods to many other metalloenzymes [72–77]. Most recently, we have studied the mechanism of CO oxidation
catalyzed by Mo–Cu carbon monoxide dehydrogenase (Mo–Cu CODH) using the
ONIOM QM/MM method [78]. The QM/MM enzyme model was built from a crystal
structure (PDB code 1N5W) [79]. Over the past two decades, the mechanism of the