Chemical modification and site-directed mutagenesis of conserved HXXH and PP-loop motif arginines and histidines in the murine bifunctional ATP sulfurylase/adenosine 5 '-phosphosulfate kinase

The sulfurylase domain of the mouse bifunctional enzyme ATP sulfurylase/ade nosine 5'-phosphosulfate (APS) kinase contains HXXH and PP-loop motifs. To elucidate the functional importance of these motifs and of conserved argini nes and histidines, chemical modification and site-directed mutagenesis stu dies were performed. Chemical modification of arginines and histidines with phenylglyoxal and diethyl pyrocarbonate, respectively, renders the enzyme inactive in sulfurylase, kinase, and overall assays. Data base searches and sequence comparison of bifunctional ATP sulfurylase/APS kinase and monofun ctional ATP sulfurylases shows a limited number of highly conserved arginin es and histidines within the sulfurylase domain. Of these conserved residue s, His-425, His-428, and Arg-421 are present within or near the HXXH motif whereas His-506, Arg-510, and Arg-522 residues are present in and around th e PP-loop, The functional role of these conserved residues was further stud ied by site-directed mutagenesis. In the HXXH motif, none of the alanine mu tants (H425A, H428A, and R421A) had sulfurylase or overall activity, wherea s they all exhibited normal kinase activity. A slight improvement in revers e sulfurylase activity (<10% residual activity) and complete restoration of forward sulfurylase was observed with R421K, Mutants designed to probe the PR-loop requirements included H506A, R510A, R522A, R522K, and D523A. Of th ese, R510A exhibited normal sulfurylase and kinase activity, R522A and R522 K showed no sulfurylase activity, and H506A had normal sulfurylase activity but produced an effect on kinase activity (<10% residual activity). The si ngle aspartate, D523A, which is part of the highly conserved GRD sequence o f the PP-loop, affected both sulfurylase and kinase activity, This mutation al analysis indicates that the HXXH motif plays a role only in the sulfuryl ase activity, whereas the PP-loop is involved in both sulfurylase and kinas e activities. Residues specific for sulfurylase activity have also been dis tinguished from those involved in kinase activity.