Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase

• Published in: Biochemistry (Easton) 1989-01, Vol.28 (2), p.471-478
• Main Authors: Huang, Shaoming, Delcamp, Tavner J, Tan, Xuehai, Smith, Philip L, Prendergast, Neal J, Freisheim, James H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Cloning, Molecular; DNA - genetics; Enzyme Stability; Enzyme-Linked Immunosorbent Assay; Humans; Kinetics; Molecular Sequence Data; Mutation; Phenylalanine; Plasmids; Spectrometry, Fluorescence; Tetrahydrofolate Dehydrogenase - genetics; Tetrahydrofolate Dehydrogenase - metabolism; Tryptophan
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00428a010

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.