| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1991: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The following results have been obtained by the present studies.
(a)Identification of an Active-Site Lysine in Leucine Dehydrogenase by Chemical Modification with Pyridoxal Phosphate
Leucine dehydrogenase from Bacillus stearothermophilus was inactivated by incubation with pyridoxal 5'-phosphate(PLP)followed by reduction with sodium borohydride. The inactivation was completely retarded in the copresence of L-leucine and NAD^+. Several PLP molecules were indeed incorporated into one mol of the enzyme subunit concomitantly with the inactivation. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the labeled protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Lys8O was most predominantly labeled and, in the presence of L-leucine and NAD^+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1 : 1 was observed between the residual activity and the amount of PLP incorporated into Lys8O. Lys8O is conserved
(b
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)Functional Analysis of Active-Site Lysine 80 in Leucine Dehydrogenase by Site-Directed Mutagenesis
Lys80 of leucine dehydrogenase has been replaced by Ala, Arg, or Gln by sitedirected mutagenesis. The Lys8O mutant enzymes purified to homogeneity showed markedly low V values(0.2-1.6% of that of the wild-type enzyme)in the oxidative deamination, but variable_<max> values in the reductive amination(Lys8O-Ala, 89% ; Lys8O-Gln, 23% ; and Lys8O-Arg, 0.4% of that of the wild-type enzyme). The Lys8O-Ala and Lys8O-Gln mutant enzymes exhibited considerably increased K_m values for alpha-keto-iso-caproate, whereas the K_m values for NAD^+ and NADH of all the mutant enzymes were similar to those of the wild-type enzyme. iso-Caproate, a non-substrate analogue of alpha-keto-iso-caproate, competitively inhibited the reaction of both the wild-type and Lys8O-Ala mutant enzymes with K_i values much higher than the K_m value for alpha-keto-iso-caproate of the wild-type enzyme, suggesting the importance of an electrostatic interaction between the *-amino group of Lys8O and the alpha-carbonyl group of alpha-keto-iso-caproate in the binding of the keto acid substrate. Exogenously added primary amines including ammonium ion remarkably accelerated the reaction catalyzed by the Lys8OAla mutant enzyme, indicating that the added amines can partially replace the function of *-amino group of Lys8O depending on both their basicities (pK_a values) and molecular volumes. These results lead to the conclusion that the *-amino group of the active site Lys8O ftinctions as a general acid-base group in the catalysis of leucine dehyarogenase. Less
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