Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

126 Chapter 6
An important point is that NADH is a stoichiometric reagent which is
bound non-covalently by the enzyme at the start of the reduction reaction
and released as NAD þ at the end of the reaction. The concentration of
NADH in solution, therefore, decreases as an enzyme-catalysed reduction
proceeds, which allows the reaction to be monitored by UV spectroscopy,
since NADH has a strong absorption at 340 nm (e ¼ 6:3 10 3 m 1 cm 1 ).
We have already met the example of horse liver alcohol dehydrogenase,
which catalyses the NAD þ -dependent oxidation of ethanol to acetaldehyde (see
Section 4.4). In a classic series of experiments by Westheimer, this enzyme was
shown to be entirely stereospeciWc for the removal of prochiral hydrogen atoms
in both the substrate and the cofactor. These experiments are illustrated in
Figure 6.5.
Incubation of enzyme with CH 3 CD 2 OH followed by re-isolation of oxidised
NAD þ revealed no deuterium incorporation into the oxidised cofactor, demonstrating
that the deuterium atom transferred to the cofactor was stereospeciWcally
removed in the reverse reaction. Isolation of the deuterium-containing
reduced cofactor (NADD) followed by chemical conversion to a substance of
established conWguration revealed that the reduced cofactor had the 4R stereochemistry.
Incubation of acetaldehyde and [4R- 2 H]-NADD with enzyme for
prolonged reaction times gave no exchange of the deuterium label into acetaldehyde,
indicating that the enzyme was also stereoselective for the removal of a
hydrogen atom from C-1 of ethanol.
Incubation with speciWcally deuteriated substrates subsequently revealed
that the enzyme removes speciWcally the proR hydrogen, which is transferred
to the C-4 proR position of NAD þ . The mechanism is thought to proceed via
direct hydride transfer, as shown in Figure 6.6. The active site of horse liver
alcohol dehydrogenase contains a Zn 2þ cofactor which activates the substrate
for nucleophilic attack. Protonation of the reduced carbonyl group is carried
out by a dyad of Ser-48 and His-51, as illustrated in Figure 6.7.
H
H
D
D
D
OH
+
N
R
CONH 2
ENZ
D
O
+
N
R
CONH 2
No D incorporation
into re-isolated NAD +
NAD +
4R- 2 H-NADD
H
O
+
H
N
R
D
CONH 2
ENZ
H D
+
OH
1R- 2 H-ethanol
N
R
CONH 2
No D incorporation
into re-isolated
CH 3 CHO
Figure 6.5 StereospeciWcity of alcohol dehydrogenase. NADD, deuterium-containing reduced
NADH.