Mutational analysis of residues in the nucleotide binding domain of human terminal deoxynucleotidyl transferase.
Human terminal deoxynucleotidyl transferase (TdT) was overexpressed in a baculovirus system. The pure recombinant enzyme was identical in size, activity, kinetic constants, and metal effects to native enzyme. Three amino acids, within either the putative nucleotide binding domain and part of a DNA polymerase consensus sequence, YGDTDSLF, or a TdT consensus sequence, GGFRRGK, were altered by site-directed mutagenesis. The four mutant forms of terminal transferase were also overexpressed in the baculovirus expression system and purified from Trichoplusia ni larvae by a monoclonal antibody affinity column and compared with wild-type enzyme with respect to thermostabilities, secondary structure, metal effects, and kinetic parameters. Three of the four mutants retained 3-16% of wild-type activity under varying metal conditions, and one of the mutants, D343E, consistently exhibited less than 0.2% of wild-type TdT activity with dATP and no activity with dGTP. All mutants had alterations in the Km for dATP. Variations in Km for dGTP were not as consistent. The Km for the other substrate, DNA initiator (dA)50) in the presence of dATP remained essentially the same as that of wild-type TdT for all mutants except D343E. The enzyme activity of all mutants was stimulated by Zn2+ at low concentrations, and this effect was diminished and reversed at higher concentrations of ZnSO4. All mutants still retained significant amounts of the secondary structure as measured by circular dichroism. These results indicated that the aspartic acid residue at position 343 is located at or near the active site and is critical for the nucleotide binding and catalytic activity.
Yang
B
B
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
Gathy
K N
KN
Coleman
M S
MS
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D004253:DNA Nucleotidylexotransferase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase