Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis
Biochemistry & Molecular Pharmacology
Graduate School of Biomedical Sciences; Department of Pharmacology
Medical Subject Headings
Alleles; Amino Acid Sequence; *Aspartic Acid; Bacteriophage T4; Cloning, Molecular; *Cysteine; Deoxyuracil Nucleotides; Escherichia coli; Hydrogen Bonding; *Hydroxymethyl and Formyl Transferases; Kinetics; *Mutagenesis, Site-Directed; Recombinant Proteins; Transferases
Life Sciences | Medicine and Health Sciences
The proposed roles of Cys148 and Asp179 in deoxycytidylate (dCMP) hydroxymethylase (CH) have been tested using site-directed mutagenesis. CH catalyzes the formation of 5-(hydroxymethyl)-dCMP, essential for DNA synthesis in phage T4, from dCMP and methylenetetrahydrofolate. CH resembles thymidylate synthase (TS), an enzyme of known three-dimensional structure, in both amino acid sequence and the reaction catalyzed. Conversion of Cys148 to Asp, Gly, or Ser decreases CH activity at least 10(5)-fold, consistent with a nucleophilic role for Cys148 (analogous to the catalytic Cys residue in TS). In crystalline TS, hydrogen bonds connect O4 and N3 of the substrate dUMP to the side-chain amide of an Asn; the corresponding residue in CH is Asp179. Conversion of Asp179 to Asn reduces the value of kcat/KM for dCMP by (1.5 x 10(4))-fold and increases the value of kcat/KM for dUMP by 60-fold; as a result, CH(D179N) has a slight preference for dUMP. Wild-type CH and CH(D179N) are covalently inactivated by 5-fluoro-dUMP, a mechanism-based inactivator of TS. Asp179 is proposed to stabilize covalent catalytic intermediates, by protonating N3 of the pyrimidine-CH adduct.
Rights and Permissions
Citation: Biochemistry. 1992 Oct 27;31(42):10315-21.
Graves, Karen Lorraine; Butler, Michelle; and Hardy, Larry W., "Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis" (1992). GSBS Student Publications. 409.