Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase of the DExH box family

Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA- dependent ATPase that serves as a transcription termination factor during v iral mRNA synthesis. NPW-I is a member of the DExH box family of nucleic ac id-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contrib utions of individual amino acids (underlined) in motifs I (GxGKT), II (DExH N), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine sc anning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142 , His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clar ified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relatio nships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent.