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Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase.
- Source :
-
Biochemistry [Biochemistry] 1989 May 30; Vol. 28 (11), pp. 4645-50. - Publication Year :
- 1989
-
Abstract
- Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)
- Subjects :
- Binding Sites
Folic Acid analogs & derivatives
Folic Acid pharmacology
Humans
Kinetics
Leucine physiology
Methotrexate pharmacology
Mutation
NADP metabolism
Phenylalanine physiology
Recombinant Proteins metabolism
Structure-Activity Relationship
Tetrahydrofolate Dehydrogenase genetics
Trimethoprim pharmacology
X-Ray Diffraction
Leucine metabolism
Phenylalanine metabolism
Tetrahydrofolate Dehydrogenase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 28
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 2765506
- Full Text :
- https://doi.org/10.1021/bi00437a020