Kinetic Relationships between the Various Activities of the Formyl-Methenyl-Methylenetetrahydrofolate Synthetase

G. Folena Wasserman, P. A. Benkovic, M. Young, S. J. Benkovic

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Abstract

The formyl-methenyl-methylenetetrahydrofolate synthetase from chicken liver catalyzes the formation of the 10-formyl- and 5,10-methenyltetrahydrofolate cofactors via three enzymatic activities. In this report we define the kinetic relationships between the activities of this trifunctional protein. An investigation of the time course for 10-formyl cofactor synthesis by computer modeling indicates that commencing with tetrahydropteroyltriglutamate, the activities of the synthetase/cyclohydrolase couple act as separate enzymic species. In contrast, 10-formyl cofactor formation from the 5,10-methylene cofactor utilizing the dehydrogenase/cyclohydrolase couple is described by a single or interactive site model that partitions the 5,10-methenyl intermediate primarily (85%) to the 10-formyl product. An unusual characteristic of the latter coupled activities is the negligible cyclohydrolase activity toward exogenous 5,10-methenyl cofactor, which serves as substrate in the individual activity assay. This is based on (1) competitive inhibition by 5,11-methenyltetrahydrohomofolate against the 5,10-methenyl derivative in the cyclohydrolase-catalyzed hydrolysis but the absence of such inhibition in the dehydrogenase/cyclohydrolase couple and (2) a pulse-chase experiment showing the failure of chase 5,10-methenyl cofactor to dilute the 10-formyl product derived from the coupled activities. The result of this coupling is to minimize the concentration of the 5,10-methenyl species, consistent with its noninvolvement in de novo purine biosynthesis.

Original languageEnglish (US)
Pages (from-to)1005-1013
Number of pages9
JournalBiochemistry
Volume22
Issue number5
DOIs
StatePublished - 1983

All Science Journal Classification (ASJC) codes

  • Biochemistry

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