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D-Amino acid transaminase (EC 2.6.1.21), from Bacillus sp. YM-1, a thermostable
enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of
novel antimicrobial agents, catalyzes the reversible transfer of an amino group
between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate,
respectively. To explore the catalytic role of Lys-145, which binds the
coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been
mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del
Pozo, A., Pospischil, M. A., Manning, J. M., Ringe, D., Stoddard, B., Tanizawa,
K., Yoshimura, T., and Soda, K. (1990) J. Biol. Chem. 265, 22306-22312) was
compared to the wild-type enzyme for its kinetic parameters. Initial velocity
studies and partial reaction isotope exchange experiments showed that the low
activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme
with saturating substrates) is an intrinsic property, confirming that
contaminating enzymes do not account for the low activity of the K145Q mutant
enzyme. The rates of the forward reaction for both wild-type and mutant enzymes
were 30-40 times higher than the rates of the reverse reaction. KM values for
the four substrates were 10 to 100 higher for the mutant compared to the
wild-type enzyme. Whereas D-alanine is preferred over L-alanine by the wild-type
enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not
efficiently discriminate between L- and D-alanine. Both wild-type and mutant
enzymes also catalyze the slow racemization of L- and D-alanine. Proton NMR
studies showed that wild-type enzyme catalyzed a time-dependent exchange of the
C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha
proton of L-alanine; the latter slow exchange rate is the same for the C alpha
proton of both L- and D-alanine with the K145Q mutant enzyme. Thus, in addition
to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid
transaminase is involved in several other important functions, i.e. it optimizes
catalytic efficiency and it maintains stereochemical fidelity. The steady-state
kinetic results on the K145Q mutant enzyme together with the findings on the
relative racemization rates and the NMR protein exchange data suggest that an
alternate base catalyzes abstraction of the alpha proton of substrate in this
mutant D-amino acid transaminase.
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