Chemistry Reference
In-Depth Information
As pointed out above, Mg
2
þ
binding to the enzyme can either be directly through protein side chains or
peptide carbonyls (inner sphere) or by indirect interactions through metal-bound water molecules (outer sphere).
Mg
2
þ
-dependent enzymes can be divided into two general classes. First, there are those in which the enzyme binds
the magnesium
substrate complex, and usually the enzyme has little or only weak interaction with the Mg
2
þ
,its
principal binding being to the substrate. Second, there are enzymes to which Mg
2
þ
binds directly, altering the
structure of the enzyme and/or playing a catalytic role.
Mg
2
þ
binding to enzymes is relatively weak (K
a
not more than 10
5
M
1
) such that the enzyme is often isolated in
the metal-free form, and Mg
2
þ
must be added to the in vitro enzyme assay system. As pointed out earlier, the
intracellular freeMg
2
þ
concentration is about 5
e
10
3
M, so thatmostMg
2
þ
-dependent enzymes have adequate local
concentrations ofMg
2
þ
for their activity. Two factors whichmakeMg
2
þ
biochemistry difficult to carry out are that the
metal is, like Zn
2
þ
, spectroscopically silent, and second that, since 1990 the only practically useful isotope
28
Mg, with
high energy
emission and a half-life of 21.3 h, has become outrageously expensive ($30,000 per mCi), such
that it is no longer used for transport studies. These practical problems may, in part, be resolved by substituting Mn
2
þ
for Mg
2
þ
to carry out spectroscopic studies, and to use substitute isotopes such as
63
Ni
2
þ
for transport studies.
b
and
g
PHOSPHORYL GROUP TRANSFER KINASES
Phosphoryl group transfer reactions either add or remove phosphoryl groups to or from cellular metabolites and
macromolecules, and play a major role in biochemistry. Phosphoryl transfer is the most common enzymatic
function coded by the yeast genome, and in addition to its importance in intermediary metabolism (Chapter 5) the
reaction is catalysed by a large number of central regulatory enzymes which often are part of signalling cascades,
such as protein kinases, protein phosphatases, ATPases, and GTPases.
Kinases are Nature's tools for introducing phosphoryl groups into organic molecules, whether they are
metabolites such as glucose and fructose-6-phosphate in the glycolysis pathway or proteins which are part of
signalling cascades, such as that which activates glycogenolysis and simultaneously inhibits glycogen synthesis
via phosphorylation of protein side chains (serine residues in this particular case). The donor of the phosphoryl
group is usually Mg
2
þ
e
ATP.
The resting adult human brain consumes around 80 mg of glucose and 50 ml of O
2
per minute, and once the
glucose has been transported across the plasma membrane it is rapidly phosphorylated by hexokinase, the first
enzyme of the glycolytic pathway. Hexokinase catalyses the transfer of a phosphoryl group from Mg
2
þ
e
ATP t o
glucose to form glucose-6-phosphate and Mg
2
þ
-ADP. It is a member of a superfamily of proteins with a common
characteristic
-fold, which is repeated in both the N-terminal and the C-terminal domains. The
members have a common ATPase domain, and include kinases, which phosphorylate not only sugars but also
glycerol, acetate, and other carboxylic acids. As illustrated by glucose binding to hexokinase (
Figure 10.2
)
,
catalysis by these enzymes is known to be accompanied by a large conformational change, which is associated
bbbababa
(a)
(b)
FIGURE 10.2
(a) yeast hexokinase; (b) in its complex with glucose.
(From
Voet & Voet, 2004
:
pp. 1591. Copyright 2004 with permission
from John Wiley and Sons, Inc.)
