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Enzymatic Hydrolysis and Group Transfer Reactions 103
Mechanism A − dissociative
RO
O
P O −
O −
O
O
RO − R'OH
+ P
ROH + − O P
− O O
−O
metaphosphate
OR'
Mechanism B − concerted
RO
O
P
O
H
O
+ R'OH RO P O ROH +
− O P OR'
− O O − R'
O −
O −
O − + R'OH ROH +
Mechanism C − associative
O
O −
RO P O −
RO P
O −
− O OH
Figure 5.28 Mechanisms for phosphoryl transfer.
OR'
− O
O
P OR'
O −
ing transfer of a proton onto the departing oxygen, and concerted phosphoryl
transfer. Phosphate diesters and triesters proceed via progressively more associative
mechanisms, either concerted (mechanism B) in the presence of a good
leaving group, or fully associative (mechanism C) in the absence of a
good leaving group.
Enzymatic hydrolysis of phosphate mono-esters is carried out by a family of
phosphatase enzymes. Of these enzymes the bovine alkaline phosphatase has
been best studied, due to its availability and its broad substrate speciWcity: it
will hydrolyse a very wide range of phosphate mono-esters. Analysis of the
stereochemistry of the alkaline phosphatase reaction using the chiral phosphate
methodology described in Section 4.4 revealed that this reaction proceeds with
retention of stereochemistry at phosphorus. Rapid kinetic analysis has revealed
that a stoichiometric amount of the alcohol product ROH is released prior to
phosphate release, and that the k cat for a range of substrates is independent of
the nature of ROH. These data suggest the existence of a phospho–enzyme
intermediate, whose breakdown is rate-determining. Incubation of enzyme with
RO 32 PO 2 3 gave enzyme labelled with 32 P, which upon tryptic digestion and
sequencing was found to be localised on a unique serine residue, Ser-102. The
active site of alkaline phosphatase, shown in Figure 5.29, contains two Zn 2þ
ions, with a separation of 3.9 Å. One zinc centre is used to bind the phosphate
mono-ester substrate, the other to activate Ser-102 for nucleophilic attack, as
shown in Figure 5.30. The phosphate oxygens are co-ordinated by Arg-166,
which provides transition state stabilisation for the phosphotransfer reaction.