Direct transfer of NADH

Direct transfer of NADH

ABSTRACT Following the criticism by Chock and Gutfreund
[Chock, P. B. & Gutfreund, H. (1988)Proc. Nall. Acad.
Sci. USA 85, 8870-8874], that our proposal of direct transfer
of NADH between glycerol-3-phosphate dehydrogenase (aglycerol
phosphate dehydrogenase, a-GDH; EC 1.1.1.8) and
L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a
misinterpretation of the kinetic data, we have reinvestigated
the transfer mechanism between this enzyme pair. By using the
“enzyme buffering” steady-state kinetic technique [Srivastava,
D. K. & Bernhard, S. A. (1984) Biochemistry 23, 4538-
45451, we examined the mechanism (random diffusion vs.
direct transfer) of transfer of NADH between rabbit muscle
a-GDH and pig heart LDH. The steady-state data reveal that
the LDH-NADH complex and the a-GDH-NADH complex can
serve as substrate for the a-GDH-catalyzed reaction and the
LDH-catalyzed reaction, respectively. This is consistent with
the direct-transfer mechanism and inconsistent with a mechanism
in which free NADH is the only competent substrate for
either enzyme-catalyzed reaction. The discrepancy between
this conclusion and that of Chock and Gutfreund comes from
(i) their incorrect measurement of the K. for NADH in the
a-GDH-catalyzed reaction, (a) inadequate design and range of
the steady-state kinetic experiments, and (ii) their qualitative
assessment of the prediction of the direct-transfer mechanism.
Our transient kinetic measurements for the transfer of NADH
from a-GDH to LDH and from LDH to a-GDH show that both
are slower than predicted on the basis of free equilibration of
NADH through the aqueous environment. The decrease in the
rate of equilibration of NADH between a-GDH and LDH
provides no support for the random-diffusion mechanism;
rather, it suggests a direct interaction between enzymes that
modulates the transfer rate of NADH. Thus, contrary to Chock
and Gutfreund’s conclusion, all our experimental data compel
us to propose, once again, that NADH is transferred directly
between the sites of a-GDH and LDH…

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