Page 109 - Color Atlas of Biochemistry

P. 109

100 Metabolism

Lactate dehydrogenase: and coenzyme, so that water molecules are
mechanism largely excluded during the electron transfer.
We can now look at the partial reactions
The principles of enzyme catalysis discussed involved in LDH-catalyzed pyruvate reduc-
on p. 90 can be illustrated using the reaction tion.
mechanism of lactate dehydrogenase (LDH)
as an example. In thefreeenzyme, His195 is protonated
(1). This form of the enzyme is therefore de-
+
scribed as E H .The coenzyme NADH is
A. Lactate dehydrogenase: catalytic cycle bound first (2), followed by pyruvate (3). It
LDH catalyzes the transfer of hydride ions (see is important that the carbonyl group of the
+
p. 32) from lactate to NAD or from NADH to pyruvate in theenzymeand theactivesitein
pyruvate. the nicotinamide ring of the coenzyme should
have a fairly optimal position in relation to
+
L-lactate + NAD ↔pyruvate + NADH + H + each other, and that this orientation should
become fixed (proximity and orientation of the
The equilibrium of the reaction strongly fa- substrates). The 98–111 loop now closes over
vors lactate formation.Athigh concentrations theactivecenter. This produces a marked
+
of lactate and NAD , however, oxidation of decrease in polarity, which makes it easier
lactateto pyruvateis also possible(see to achieve the transition state (4; water ex-
p.18). LDH catalyzes the reaction in both di- clusion). In the transition state, a hydride ion,
–
rections, but—like all enzymes—it has no ef- H (see p. 32), is transferred from the coen-
fect on chemical equilibrium. zyme to the carbonyl carbon (group transfer).
As the reaction is reversible, the catalytic The transient—and energetically unfavora-
process can be represented as a closed loop. ble—negative charge on the oxygen that oc-
The catalytic cycle of LDH is reduced to six curs here is stabilized by electrostatic inter-
“snapshots” here. Intermediate steps in catal- action with Arg-109 (stabilization of the tran-
ysis such as thoseshown here areextremely sition state). At thesametime, a proton from
short-lived and therefore dif cult to detect. His-195 is transferred to this oxygen atom
Their existence was deduced indirectly from a (group transfer), giving rise to the enzyme-
+
large number of experimental findings—e. g., bound products lactate and NAD (5). After
kinetic and binding measurements. the loop opens, lactate dissociates from the
Many amino acid residues play a role in the enzyme, and the temporarily uncharged imi-
active center of LDH. They can mediate the dazole group in His-195 again binds a proton
binding of the substrate and coenzyme, or from the surrounding water (6). Finally, the
+
take part in one of the steps in the catalytic oxidized coenzyme NAD is released, and the
cycle directly. Only the side chains of three initial state (1) is restored. As the diagram
particularly important residues are shown shows, the proton that appears in the reaction
+
here. The positively charged guanidinium equation (NADH + H ) is not bound together
group of arginine-171 binds the carboxylate with NADH, but after release of the lacta-
group of the substrate by electrostatic inter- te—i. e., between steps (5)and (6)of the
action. The imidazole group of histidine-195 is previous cycle.
involved in acid–base catalysis, and the side Exactly the same steps occur during the
chain of arginine-109 is important for the sta- oxidation of lactate to pyruvate, but in the
bilization of the transition state. In contrast to opposite direction. As mentioned earlier, the
His-195, which changes its charge during cat- direction which the reaction takes depends
alysis, the two essential arginine residues are not on the enzyme, but on the equilibrium
constantly protonated. In addition to these state—i. e., on the concentrations of all the
three residues, the peptide loop 98–111 men- reactants and the pH value (see p.18).
tioned on p. 98 is also shown here schemati-
cally (red). Its function consists of closing the
active center after binding of the substrate

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