 |
|
Title
|
|
N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis.
|
|
|
Authors
|
|
A.A.Hoskins,
M.Morar,
T.J.Kappock,
I.I.Mathews,
J.B.Zaugg,
T.E.Barder,
P.Peng,
A.Okamoto,
S.E.Ealick,
J.Stubbe.
|
|
|
Ref.
|
|
Biochemistry, 2007,
46,
2842-2855.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from
Escherichia coli catalyzes the reversible interconversion of N5-CAIR to
carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer.
Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray
crystallography together provide new insight into the mechanism of this unusual
transformation. These studies suggest that a conserved, protonated histidine
(His45) plays an essential role in catalysis. The importance of proton transfers
is supported by DFT calculations on CAIR and N5-CAIR analogues in which the
ribose 5'-phosphate is replaced with a methyl group. The calculations suggest
that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in
energy than its aromatic counterpart, implicating this species as a potential
intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE
cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR
analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been
determined and provide the first insight into the binding of an intact PurE
substrate. A comparison of 19 available structures of PurE and PurE mutants in
apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2
binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the
carboxylate or CO2 interacts with backbone amides. This work has led to a
mechanistic proposal in which the carboxylate orients the substrate for proton
transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole
ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the
aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate
isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.
|
|
|
|