MECHANISMS OF ENZYMATIC REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 62

electrostatically with of AMP-PNP. The hydroxyl oxygen of and

oxygens of and of bound MgAMP-PNP are directly liganded to the

ion. Carboxylate oxygens βGlu192 and βAsp256 interact with through water

molecules. The of probably plays a key role in the catalysis. This

group locates 4.4 from the of MgAMP-PNP, where it is hydrogen bonding

to a water molecule. Modification of by dicyclohexylcarbodiimid accompanied
inactivation of According to the suggestion of Abraham et al., (1994), this
group functions as a general base that activates the water molecule for an attack on the
during ATP hydrolysis, while the guanidinum of

Å

might stabilizes a

pentavalent phosphorus transition state during catalysis.
The following pathway having a large H-bonds polarization in hydrated subunits has
been proposed (Zundel, 2000): carboxylate Ala79, Tyr10 (c-subunit), Glu219, His245 (a),
Asp61(c), Arg41 (c) and Arg 210 (a). A proton conducts by the mechanism of concerted
proton tunneling within less than picosecond. Chemical modification and mutagenesis
studies implicate that of is involved in the catalysis, most probably

indirectly (Weber and Senior, 1997).
It has been proposed (Likhtenshtein and Shilov, 1976; Likhtenshtein, 1988a) that the
first result of ATP hydrolysis in the active site of energy-converting enzymes may be the
forced protonation of one of the functional groups of the active site X followed by the
formation of the protonated energy-reach intermediate The energy of which is
not in equilibrium with the environment, may be then utilized for performance of
chemical, mechanical or electrical work. Such a mechanism will be efficient if this
intermediate is shielded from the water environment long enough for the performance of
work. A similar idea was exposed by Williams (1982) who suggested that protons are
generated in the vicinity of the ATP-synthetase by oxidative or photon-energy flow
through the ATP-synthase site without equilibrating with the bulk phases.
As far as concern the mechanism of ATP hydrolysis, the nucleophilic capacity of the
group is not sufficient for fast cleavage of the bond of
ATP. A more realistic explanation of the process is that the attack of water molecule on
the bond results in the force protonation of this carboxylic group accounting for the energy
released in the ATP hydrolysis. Protonation annihilates the carboxylate negative charge.
The formation of such an nonequilibrium intermediate violates the electrostatic balance in
the active site and can induce conformational transition favorable for a series of proton
jumps from the energetically nonequilibrium group along the translocation

channel.
Recently a mechanism that links conformational coupling of energetics of two
chemical reactions through conformational change during a catalyst reaction cycle was
proposed (Leyh, 1999). ATP sulfurylase from E. coli catalyzes and energetically links the
hydrolysis of GTP and the synthesis of activated sulfate, APS (adenosine-5'-
phosphosulfate) by reaction between ATP and sulfate. Experiments showed that the
enzyme undergoes a conformational change in the GTP-binding reaction and the rate-
limiting conformational step precedes the GTP hydrolysis. Formation of active signaling
conformation promotes synthesis of APS. Active conformation is transformed to inactive
during the release of Pi and

Existing structural data don't contradict the