| Project/Area Number |
05680522
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | TOYAMA MEDICAL AND PHARMA CEUTICAL UNIVERSITY |
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Principal Investigator |
GOMI Tomoharu Toyama Medical and Pharmaceutical University, Scientific Instrument Center, Associate Professor, 実験実習機器センター, 助教授 (40135033)
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| Co-Investigator(Kenkyū-buntansha) |
FUJIOKA Motoji Toyama Medical and Pharmaceutical University, Faculty of Medicine, Professor, 医学部, 教授 (30030000)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1994: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Methyltransferase / S-Adenosylmethionine / Guanidinoacetate methyltransferase / Glycine methyltransferase / Affinity labeling / Site-directed mutagenesis / Positive cooperativity / Amino-terminal acetylation |
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| Research Abstract |
1. Guanidinoacetate methyltransferase (GAMT) : (1) Tyr-136 that is photoaffinity -labeled by AdoMet resides in a region whose structural feature is shared by most mammalian methyltransferases (mMTs). Amino acid replacements were introduced to the region. The results of precise kinetic analyzes of mutant enzymes indicate that Asp-134 is crucial for binding AdoMet. (2) We found that mMTs share a sequence motif similar to one that is common in nucleotide-binding proteins. Studies by site-directed mutagenesis suggested the importance of the motif for the enzymatic activity of GAMT.
2. Glycine methyltransferase (GMT) : (1) It is reported that rat GMT shows positive cooperativity toward AdoMet while rabbit enzyme dose not. Cloning and sequencing of GMTs from rabbit, human, and pig livers charified that all GMTs including rat enzyme have very similar structures, and kinetic analyzes with liver extracts revealed that they all exhibit the cooperativity toward AdoMet. (2) The recombinant rabbit GMT did not show the kinetic cooperativity. The only structural difference between recombinant-and liver enzyme was that the amino-terminal Val residue of the former is free while that of the latter is acetylated.This cbservation and the result of pH study suggest that the acetylation confers on GMT the cooperativity toward AdoMet by masking amino-terminal positive charge. (3) Recombinant rat GMT was crystallized and preliminary X-ray diffraction data set was obtained.
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