MECHANISMS OF ENZYMATIC REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 61

soluble part, composed of five different subunits and the membrane
bilayer integrated part made up of three different subunits a, b (2), and c (11). There are
six nucleotide-binding sites on the enzyme: three catalytic sites, located on b-subunits, and
three noncatalytic sites, located on a-subunits.
According to the binding change model proposed by Boyer (2001 and references
therein) the ATP synthesis proceeds in the following stages: (1) condensation of ADP with
phosphate (Pi) that occurs inside the enzyme without energy input from proton
translocation, (2) sequential operation of three catalytic sites of F , which have tight,

loose, and open conformation and undergo binding, interconversion and release steps of
the process, respectively, and (3) binding of ADP and Pi to a low -affinity catalytic site
that promote release of ATP bound to a high-affinity catalytic site for the expense of
energy provided by proton translocation. Boyer postulated also that the enzyme operates
by a rotational mechanism in which proton translocation in the portion drives an internal
rotation of of F 1 , causing sequential conformational change in the
Elucidation of the crystal structure of the bovine heart mitochondrial
(Abraham et al., 1994, Gibbons et. al., 2000) focused attention on rotational catalysis in
coupling ATP synthesis and hydrolysis with the proton translocation. Electron microscopy
and X-ray structural analysis studies have shown that theF 1 (part of the enzyme is separated
from the by a narrow stalk of around 45 Å.
In a series of elegant biochemical and chemical engineering works, direct evidence for
rotation of c-ring ang relative to during catalysis were presented. A
mutation allowed Duncan et al., (1995) to induce formation of a specific disulfide bond
between and in soluble from E. coli. Formation of the crosslink inactivated the
enzyme, and reduction restored full activity. In contrast, fixing to by
cross-linking does not greatly impair either the ATPase activity or coupling proton
translocation Counterclockwise rotation of a fluorescently-labeled actin filament attached
to the of driven by ATP hydrolysis was directly demonstrated with
the use of a fluorescence microscope (Noji et al., 2001 and references therein).
Investigation of kinetics of the catalytic process revealed drastic differences in the
values for reaction in the presence of substoichiometric concentrations of substrate,
MgATP, occurring in lower than that in saturating conditions (Allison, 1998).
This result clearly indicates strong positive cooperativity of the process. The Allison
models for the minimal steps of ATP hydrolysis and synthesis under saturating conditions
suggest that catalytic site

1

adopt only two stable conformations, rather than three

postulated by Boyer.
Recently new models were proposed and animated to demonstrate how each of
subunit pairs can be stabilized against rotation of the

while also maintaining the

chemical equivalency of the three pairs (Blum et al., 2000).
For elucidation of chemical mechanisms of ATP hydrolysis and synthesis and proton
translocation positions of the enzyme groups in the vicinity of the binding substrate,
MgAMP-PNP (AMN-PNP is 5'-adenylyl-imidodiphosphate) and MgADP, are of special
interest (Abrahams et al., 1994, Allison, 1998). In the liganding catalytic sites the adenine
of bound MgAMP-PNP,. and MgADP, is present in a hydrophobic pocket
contributed by two Phe, Tyr and Val. In this state,

of interacts