Role of Lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

Journal Article · Tue Sep 04 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00487a011· OSTI ID:5763078

Huang, Shaoming; Tan, Xuehai; Thompson, P D; Freisheim, J H ^[1]; Appleman, J R; Blakley, R L ^[2]; Sheridan, R P; Venkataraghavan, R ^[3]

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1. Medical College of Ohio, Todedo (USA)
2. St. Jude Children's Research Hospital, Memphis, TN (USA)
3. Lederle Laboratories, Pearl River, NY (USA)

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. 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.

• OSTI ID: 5763078
• Journal Information: Biochemistry; (USA), Vol. 29:35; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LYSINE
BIOCHEMISTRY
OXIDOREDUCTASES
BIOCHEMICAL REACTION KINETICS
PEPTIDES
ENZYME IMMUNOASSAY
BIOLOGICAL PATHWAYS
COENZYMES
ENDONUCLEASES
GLYCINE
NADP
PH VALUE
SULFUR 35
AMINO ACIDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
CARBOXYLIC ACIDS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DNA-ASE
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
HYDROLASES
IMMUNOASSAY
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SULFUR ISOTOPES
550201* - Biochemistry- Tracer Techniques