Roles of Cys148 and Asp179 in catalysis by deoxycytidylate hydroxymethylase from bacteriophage T4 examined by site-directed mutagenesis.
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.
Graves
K L
KL
Department of Pharmacology, University of Massachusetts Medical Center, North Worcester 01655.
Butler
M M
MM
Hardy
L W
LW
D000483:Alleles; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003545:Cysteine; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014166:Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; C007267:2'-deoxyuridylic acid; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; C100226:deoxycytidylate hydroxymethyltransferase; D003545:Cysteine