Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis

• Published in: Biochemistry (Easton) 1992-10, Vol.31 (42), p.10315-10321
• Main Authors: Graves, Karen L, Butler, Michelle M, Hardy, Larry W
• Format: Article
• Language: English
• Subjects: Alleles; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Aspartic Acid; Bacteriophage T4 - enzymology; Bacteriophage T4 - genetics; Biological and medical sciences; Cloning, Molecular; Cysteine; Deoxyuracil Nucleotides - metabolism; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hydrogen Bonding; Hydroxymethyl and Formyl Transferases; Kinetics; Mutagenesis, Site-Directed; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Transferases; Transferases - genetics; Transferases - isolation & purification; Transferases - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00157a020

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.