Studies on identifying the binding sites of folate and its derivatives in Lactobacillus casei thymidylate synthase
It was shown that folate and its derivatives have a profound effect on stabilizing thymidylate synthase in vitro and in vivo, as a consequence of ternary formation between the folate, dUMP, or FdUMP, and the synthase. The degree to which complex formation is affected can be revealed qualitatively by circular dichroism and quantitatively by equilibrium dialysis using the Lactobacillus casei synthase. In contrast to the pteroylmonoglutamates, the pteroylpolyglutamates bind to thymidylate synthase in the absence of dUMP, but even their binding affinity is increased greatly by this nucleotide or its analogues. Similarly, treatment of the synthase with carboxypeptidase A prevents the binding of the pteroylmonoglutamates and reduces the binding of the polyglutamates without affecting dUMP binding. The latter does not protect against carboxypeptidase inactivation but does potentiate the protective effect of the pteroylpolyglutamates. To determine the region of the synthase involved in the binding of the glutamate residues, Pte(/sup 14/C)GluGlu6 was activated by a water soluble carbodiimide in the presence and absence of dUMP. This folate derivative behaved as a competitive inhibitor of 5,10-CH/sub 2/H/sub 4/PteGlu, in contrast to methotrexate which was non-competitive. Separation of the five cyanogen bromide peptides from the L. casei synthase revealed 80% of the radioactivity to be associated with CNBr-2 and about 15% with CNBr-4. Chymotrypsin treatment of CNBr-2 yielded two /sup 14/C-labeled peaks on high performance liquid chromatography, with the slower migrating one being separated further into two peaks by Bio-gel P2 chromatography. All three peptides came from the same region of CNBr-2, encompassing residues 47-61 of the enzyme. From these studies it would appear that the residues most probably involved in the fixation of PteGlu7 are lysines 50 and 58. In contrast, methotrexate appeared to bind to another region of CNBr-2.
- Research Organization:
- Division of Laboratories and Research, New York State Department of Health, Albany
- OSTI ID:
- 5552237
- Journal Information:
- Adv. Exp. Med. Biol.; (United States), Vol. 163
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
FOLIC ACID
METABOLISM
LIGASES
BIOCHEMICAL REACTION KINETICS
MOLECULAR STRUCTURE
PEPTIDES
LIQUID COLUMN CHROMATOGRAPHY
CARBON 14 COMPOUNDS
CARBOXYPEPTIDASES
CHYMOTRYPSIN
DICHROISM
METHOTREXATE
THYMINE
TRACER TECHNIQUES
AMINO ACIDS
ANTIMETABOLITES
AROMATICS
AZAARENES
AZINES
CARBOXYLIC ACIDS
CHROMATOGRAPHY
DRUGS
ENZYMES
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
PTERIDINES
PYRIMIDINES
REACTION KINETICS
SEPARATION PROCESSES
SERINE PROTEINASES
URACILS
VITAMIN B GROUP
VITAMINS
550200* - Biochemistry
560300 - Chemicals Metabolism & Toxicology