Lack of functional significance of Cys227 and Cys234 in terminal deoxynucleotidyltransferase.
Identification of the three functional regions (catalytic, nucleotide substrate-binding, DNA substrate-binding) of the monofunctional template independent DNA polymerase terminal deoxynucleotidyltransferase has not been completely established. The potential participation of 2 amino acid residues, Cys227 and Cys234, has been controversial, and conflicting data have been published. To investigate the role of Cys227, the human terminal transferase cDNA was modified by site-directed mutagenesis to introduce a glycine codon at this position. Mutant and control wild-type human terminal transferase cDNAs had to be inserted into baculovirus genomes by homologous recombination and overexpressed in Trichoplusia ni insect larvae because terminal transferase cDNAs have not been successfully expressed in bacterial systems. The Cys227----Gly mutant and wild-type enzymes displayed similar km values for both the nucleotide (dGTP) and DNA initiator (dA50) substrates. The kcat for the mutant enzyme (0.56 s-1) was comparable to that of the native enzyme (0.58 s-1). Additionally, catalysis by both mutant and wild-type enzymes was stimulated by Zn2+. These results together with the observation that the amino acid residue at position 234 is not conserved across species indicated that neither Cys234 nor Cys227 is an essential residue in the active site of terminal transferase.
Medin
J A
JA
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
Coleman
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D003545:Cysteine; D004253:DNA Nucleotidylexotransferase / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D051379:Mice; D015183:Restriction Mapping; D013816:Thermodynamics; D014162:Transfection
D004253:DNA Nucleotidylexotransferase; D003545:Cysteine