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Role of lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase
- Source :
- Biochemistry. 29(35)
- Publication Year :
- 1990
-
Abstract
- Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2{prime}-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of K{sub m} values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating K{sub m} and k{sub cat} values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The ratio of K{sub m}(NADH)/K{sub m}(NADPH) decreases from 69 in the wild-type enzyme to 4.7 in the K54Q enzyme, suggesting that Lys-54, among other interactions between protein side-chain residues and the 2{prime}-phosphate, makes a major contribution in terms of binding energy and differentiation of K{sub m} values for NADPH and NADH.more » Agents at concentrations that show activating effects on the wild-type enzyme such as potassium chloride and urea all inactivate the K54Q enzyme. There appear to be no gross conformational differences between wild-type and K54Q enzyme molecules as judged by competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase and from protease susceptibility studies on both wild-type and K54Q mutant enzymes. The pH-rate profiles using NADPH for K54Q and wild-type enzymes show divergences at certain pH values, suggesting the possibility of alteration(s) in the steps of the catalytic pathway for the K54Q enzyme.« less
- Subjects :
- Models, Molecular
Stereochemistry
Recombinant Fusion Proteins
Lysine
Mutant
Molecular Sequence Data
Biochemistry
Cofactor
Biological pathway
Dihydrofolate reductase
Humans
Nucleotide
Amino Acid Sequence
chemistry.chemical_classification
biology
Base Sequence
Hydrogen-Ion Concentration
NAD
Amino acid
Kinetics
Tetrahydrofolate Dehydrogenase
Enzyme
chemistry
biology.protein
Mutagenesis, Site-Directed
Oligonucleotide Probes
NADP
Subjects
Details
- ISSN :
- 00062960
- Volume :
- 29
- Issue :
- 35
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....68fb0e24c287d6e81b27364a7ba67f53