Pl. Cummins et Je. Gready, Energetically most likely substrate and active-site protonation sites and pathways in the catalytic mechanism of dihydrofolate reductase, J AM CHEM S, 123(15), 2001, pp. 3418-3428
Despite much experimental and computational study, key aspects of the mecha
nism of reduction of dihydrofolate (DHF) by dihydrofolate reductase (DHFR)
remain unresolved, while the secondary DHFR-catalyzed reduction of folate h
as been little studied. Major differences between proposed DHF mechanisms a
re whether the carboxylate group of the conserved active-site Asp or Glu re
sidue is protonated or ionized during the reaction, and whether there is di
rect protonation of N5 or a proton shuttle from an initially protonated car
boxylate group via O4. We have addressed these questions for both reduction
steps with a comprehensive set of ab initio quantum chemical calculations
on active site fragment complexes, including the carboxyl side chain and, p
rogressively, all other polar active-site residue groups including conserve
d water molecules. Addition of two protons in two steps was considered. The
polarization effects of the remainder of the enzyme system were approximat
ed by a dielectric continuum self-consistent reaction field (SCRF) model us
ing an effective dielectric constant (epsilon) of 2. Optimized geometries w
ere calculated using the density functional (B3LYP) method and Onsager SCRF
model with the 6-31G* basis. Single-point energy calculations were then ca
rried out at the B3LYP/6-311+G** level with either the Onsager or dielectri
c polarizable continuum model. Additional checking calculations at MP2 and
HF levels, or with other basis sets or values of E, were also done. From th
e results, the conserved water molecule, corresponding to W206 in the E, co
li DHFR complexes, that is H-bonded to both the OD2 oxygen atom of the carb
oxyl (Asp) side chain and O4 of the pterin/dihydropterin ring, appears crit
ically important and may determine the protonation site for the enzyme-boun
d substrates. In the absence of W206, the most stable monoprotonated specie
s are the neutral-pair I-enol forms of substrates with the carboxyl group O
D2 oxygen protonated and H-bonded to N3. If W206 is included, then the most
stable forms are still the neutral-pair complexes but now for the N3-H ket
o forms with the protonated OD2 atom H-bonding with W206. A second proton a
ddition to these complexes gives protonations at N8 (folate) or N5 (DHF), C
alculated H-bond distances correlate well with those for the conserved W206
observed in many X-ray structures. For all structures with occluded M20 lo
op conformations (closed active site), OD2-N3 distances are less than OD2-N
A2 distances, which is consistent with those calculated for protonated OD2
complexes. Thus. the results (B3LYP; epsilon = 2 calculations) support a me
chanism for both folate and THF reduction in which the OD2 carboxyl oxygen
is first protonated, followed by a direct protonation at N8 (folate) and N5
(DHF) to obtain the active cation complexes, i.e., doubly protonated. The
results do not support a proposed protonated carboxyl with DHF in the enol
form for the Michaelis complex, nor an ionized carboxyl with protonated eno
l-DHF as a catalytic intermediate. However, as additional calculations for
the monoprotonated complete complexes show a reduction in the energy differ
ences between the neutral-pair keto and ion-pair keto (N8- or NS-protonated
) forms, we are extending the treatment using combined quantum mechanics an
d molecular mechanics (QM/MM) and molecular dynamics simulation methods to
refine the description of the protein/solvent environment and prediction of
the relative stabilization free energies of the various (OD2, O4, N5, and
N8) protonation sites.