Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Kinetic and Chemical Mechanism of Pig Liver Methenyltetrahydrofolate Synthetase

돼지 간 유래의 methenyltetrahydrofolate synthetase 반응메커니즘

  • Cho, Yong-Kweon (Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University)
  • 조용권 (창원대학교 자연과학대학 보건의과학과)
  • Published : 2008.08.30
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Abstract

Methenyltetrahydrofolate synthetase extract was obtained from mouse liver and purified via $30{\sim}70%$ ammonium sulfate fractionation, Fast Q anion exchange and phenyl agarose chromatography. HPLC gel chromatography and SDS-polyacrylamide electrophoresis experiments showed that the enzyme is a monomer with molecular weight of 23 kDa. Optimum temperature and pH were $35^{\circ}C$ and 6.5, respectively. The enzyme was chemically modified only by tetranitromethane and 1-ethyl-3- (3-dimethyl aminopropyl)-carbodiimide (EDC), indicating that tyrosine and carboxylate are in the active site. pH studies showed that 2 tyrosines are involved in the binding of the substrates and a carboxylate in catalysis. Therefore, the chemical mechanism of the enzyme is likely that 2 tyrosines bind to ATP and 5-formylTHFand a carboxylate acts as a general base.

$30{\sim}70%$ 황산암모늄 분획, Fast Q 음이온 교환수지 및 Phenyl Agarose 수지를 통해 돼지간유래의 methenyltetrahydrofolate synthetase를 정제하였으며 정제도는 119이었다. SDS-PAGE 및 HPLC를 이용한 gel permeation column chromatography에 의해 이 효소는 분자량이 23 kDa인 단량체로 확인되었다. 최적 온도와 최적 pH는 각각 $35^{\circ}C$ 와 6.5이었다. 이 효소는 tetranitromethane 및 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC)에 의해서만 활성이 감소되었는데, 이는 티로신과 카르복실산이 효소의 활성부위에 존재한다는 것을 나타낸다. 활성부위의 pH 실험으로부터 2개의 티로신이 기질의 결합에 관여하며 하나의 카르복실기가 촉매반응에 관여한다는 사실이 관찰되었다. 따라서 이 효소는 2개의 티로신이 ATP와 5-formylTHF과 결합하며 하나의 카르복실기가 일반염기로서 촉매한다는 것을 알 수 있다.

Keywords

References

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