As part of an established program in pteridine biochemistry, dilhydrofolate reductase (DHFR), and dihydropteridine reductase (DHPtR), two enzymes which convert 7,8-dihydrofolate and 'quinonoid' dihydrobiopterin to their respective tetrahydro analogs, and phenylalanine hydroxylase (PAH), which converts phenylalanine to tyrosine, have each been purified to homogeneity in mg quantities from rat liver; a tissue in which the three enzymes work cooperatively. In the current proposal the purified reductases are to be examined under a variety of conditions (e.g., differing pH, ionic strengths, activators, inhibitors, etc.) for cross-specificity of substrates. These results might suggest overlapping functions for the two enzymes in vivo, an observation that would be of particular interest as DHPtR is widely distributed in cell cultures and tissues which contain no ancillary hydroxylases. The dimeric, NADH requiring DHPtR (Mr approx. 51,000) will be further characterized by interaction with fluorescent probes, radioactive photolabile substrate analogs, generation of monoclonal antibodies, by preliminary high resolution NMR analysis, sequencing, and after crystallization by X-ray techniques. Wherever structural comparisons could be informative, DHFR (Mr approx. 22,500) from the same source will also be examined. A variety of fluorescent folate analogs are to be synthesised both as aids to the above enzyme characterizations and as probes for pteridine and folate cellular uptake pathways. Additionally, of long standing interest to this laboratory have been the diverse oxidation path ways of tetrahydropteridines, therefore, to complement the enzymatic investigations, a variety of methyl-substituted tetrahydropteridines have been synthesized and are to be examined in model systems to define the factors which control their paths of degradation. As a corollary to the project methotrexate will be bound to a series of synthetic peptides to examine their properties as in vivo, site-directed carriers, with improved chemotherapeutic potential.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA011778-18
Application #
3163556
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1979-05-01
Project End
1988-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
18
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92037
Zhao, Haiyan; Bray, Tom; Ouellette, Marc et al. (2003) Structure of pteridine reductase (PTR1) from Leishmania tarentolae. Acta Crystallogr D Biol Crystallogr 59:1539-44
Chang, C F; Bray, T; Varughese, K I et al. (1999) Comparative properties of three pteridine reductases. Adv Exp Med Biol 463:403-10
Chang, C F; Bray, T; Whiteley, J M (1999) Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling. Arch Biochem Biophys 368:161-71
Kiefer, P M; Grimshaw, C E; Whiteley, J M (1997) The comparative interaction of quinonoid (6R)-dihydrobiopterin and an alternative dihydropterin substrate with wild-type and mutant rat dihydropteridine reductases. Biochemistry 36:9438-45
Wang, J; Leblanc, E; Chang, C F et al. (1997) Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1. Arch Biochem Biophys 342:197-202
Kiefer, P M; Varughese, K I; Su, Y et al. (1996) Altered structural and mechanistic properties of mutant dihydropteridine reductases. J Biol Chem 271:3437-44
Varughese, K I; Xuong, N H; Kiefer, P M et al. (1994) Structural and mechanistic characteristics of dihydropteridine reductase: a member of the Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases. Proc Natl Acad Sci U S A 91:5582-6
Varughese, K I; Xuong, N H; Whiteley, J M (1994) Structural and mechanistic implications of incorporating naturally occurring aberrant mutations of human dihydropteridine reductase into a rat model. Int J Pept Protein Res 44:278-87
Whiteley, J M; Xuong, N H; Varughese, K I (1993) Is dihydropteridine reductase an anomalous dihydrofolate reductase, a flavin-like enzyme, or a short-chain dehydrogenase? Adv Exp Med Biol 338:115-21
Varughese, K I; Su, Y; Skinner, M M et al. (1993) Two crystal structures of rat liver dihydropteridine reductase. Adv Exp Med Biol 338:123-6

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