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(Received for publication, April 5, 1996, and in revised form, April 24, 1996)
From the L-Lactate monooxygenase (LMO)
from Mycobacterium smegmatis was mutated at glycine 99 to
alanine, and the properties of the resulting mutant (referred to as
G99A) were studied. Mutant G99A of LMO was designed to test the
postulate that the smaller glycine residue in the vicinity of the
Volume 271, Number 29,
Issue of July 19, 1996
pp. 17226-17233
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,§
and
Department of Biological Chemistry,
University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 and the § Department of Veterans Affairs Medical Center, Ann
Arbor, Michigan 48105
-carbon methyl group of lactate in wild-type LMO has less steric
hindrance, leading to the retention and oxidative decarboxylation of
pyruvate in the active site, a unique property of LMO in contrast to
other members of the FMN-dependent oxidase/dehydrogenase
family. G99A has been shown to be readily reduced by
L-lactate at a rate similar to that of the wild-type
enzyme. The binding of pyruvate to reduced G99A is 4-fold weaker than
that to the wild-type enzyme. A dramatic change of this mutation is
that G99A has a much lower oxygen reactivity than the wild-type enzyme.
Pyruvate-bound reduced G99A reacts with O2 at a rate
~105-fold slower than the wild-type enzyme, and free
reduced G99A reacts with O2 at a rate ~100-fold slower
than the wild-type enzyme. Due to the very low oxygen reactivity of the
pyruvate-bound reduced enzyme, G99A has been shown to catalyze the
oxidation of L-lactate to pyruvate and hydrogen peroxide
instead of acetate, carbon dioxide, and water, the normal
decarboxylation products of pyruvate and hydrogen peroxide. Thus, the
mutation alters the enzyme from its L-lactate monooxygenase
activity to L-lactate oxidase activity. However, compared
with L-lactate oxidase, G99A has a much lower reactivity
toward oxygen. Our results also reveal that the small steric change
around N-5 of the flavin causes a profound change in the electronic
distribution in the catalytic cavity of the enzyme and imply that
electrostatic interactions in the active site provide an important
factor for control of O2 reactivity.
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