sorry, we can't preview this file
...but you can still download bi0619977_si_002.qt
bi0619977_si_002.qt (39.71 MB)
A Complete Structural Description of the Catalytic Cycle of Yeast Pyrophosphatase†,‡
dataset
posted on 2007-02-06, 00:00 authored by Esko Oksanen, Anna-Karoliina Ahonen, Heidi Tuominen, Vesa Tuominen, Reijo Lahti, Adrian Goldman, Pirkko HeikinheimoWe have determined the structures of the wild type and seven active site variants of yeast
inorganic pyrophosphatase (PPase) in the presence of Mg2+ and phosphate, providing the first complete
structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require
four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen
contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates. In the
“closed” wild-type active site, one of the two product phosphates has already dissociated, while the D115E
variant “open” conformation is of the hitherto unobserved two-phosphate and two-“bridging” water active
site. The mutations affect metal binding and the hydrogen bonding network in the active site, allowing us
to explain the effects of mutations. For instance, in Y93F, F93 binds in a cryptic hydrophobic pocket in
the absence of substrate, preserving hydrogen bonding in the active site and leading to relatively small
changes in solution properties. This is not true in the presence of substrate, when F93 is forced back into
the active site. Such subtle changes underline how low the energy barriers are between thermodynamically
favorable conformations of the enzyme. The structures also allow us to associate metal binding constants
to specific sites. Finally, the wild type and the D152E variant contain a phosphate ion adjacent to the
active site, showing for the first time how product is released through a channel of flexible cationic side
chains.