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

Abstract

The sulfurylase domain of the mouse bifunctional enzyme ATP sulfurylase/adenosine 5 -phosphosulfate (APS) kinase contains HXXH and PP-loop motifs. To elucidate the functional importance of these motifs and of conserved arginines and histidines, chemical modification and site-directed mutagenesis studies 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 monofunctional ATP sulfurylases shows a limited number of highly conserved arginines and histidines within the sulfurylase domain. Of these conserved residues, 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 the PP-loop. The functional role of these conserved residues was further studied by site-directed mutagenesis. In the HXXH motif, none of the alanine mutants (H425A, H428A, and R421A) had sulfurylase or overall activity, whereas they all exhibited normal kinase activity. A slight improvement in reverse sulfurylase activity (<10% residual activity) and complete restoration of forward sulfurylase was observed with R421K. Mutants designed to probe the PP-loop requirements included H506A, R510A, R522A, R522K, and D523A. Of these, R510A exhibited normal sulfurylase and kinase activity, R522A and R522K showed no sulfurylase activity, and H506A had normal sulfurylase activity but produced an effect on kinase activity (<10% residual activity). The single aspartate, D523A, which is part of the highly conserved GRD sequence of the PP-loop, affected both sulfurylase and kinase activity. This mutational analysis indicates that the HXXH motif plays a role only in the sulfurylase activity, whereas the PP-loop is involved in both sulfurylase and kinase activities. Residues specific for sulfurylase activity have also been distinguished from those involved in kinase activity.Recently there has been increased interest in the sulfateactivating bifunctional enzyme, ATP sulfurylase/APS 1 kinase (PAPS synthetase). With the recent cloning of two isoforms of sulfurylase/kinase (SK) from both mouse, MSK1 (1) and MSK2 (2), and human, HSK1 (3, 4) and HSK2 (3), increased efforts are directed toward understanding the structure-function relationship of the bifunctional enzyme. This enzyme was initially identified by Sugahara and Schwartz (5-7) as the site of the brachymorphic defect in mice. The enzyme was subsequently purified to homogeneity and complete kinetic, functional, and structural analyses of the two-step pathway revealed that both activities reside on a single bifunctional protein that uses a channeling mechanism to efficiently produce PAPS (8 -12). We have also reported the cloning of two mouse bifunctional enzyme isoforms, MSK1 (1) and MSK2 (2), and have shown that a point mutation in the kinase portion of MSK2 renders the enzyme inactive, causing ...

Identification and Functional Characterization of the Novel BM-motif in the Murine Phosphoadenosine Phosphosulfate (PAPS) Synthetase

“…Generation of Mutant Constructs-Our previous extensive mutational analyses (9,11) were performed on the SK1 isoform protein, thus SK1 was used for functional analysis of the BM-motif (residues Leu 86 -Lys 97 ). Site-directed mutations in SK1 cDNA were generated by insertion of double-stranded oligonucleotides.…”

“…the ATP binding (P)-loop (9), phosphoryl transfer (FISP) (10), phosphodiester-cleavage (11), and pyrophosphate binding (PP)-loop (11), all of which we have shown by mutational analysis to confer molecular binding or enzymatic activity to SK1. Similarly, sequence conservation around the bm mutation site (Gly 79 ) is very high; however, no function had been established for this region.…”

Kinetic and Stability Properties of Penicillium chrysogenum ATP Sulfurylase Missing the C-terminal Regulatory Domain

“…Residues 171-395 compose the central catalytic domain. Several residues that have been shown to be essential for activity (10,11) are located in this domain. Residues 331-389 form a small subdomain, called Domain III in the yeast structure (12,13).…”

Structure and Mechanism of Soybean ATP Sulfurylase and the Committed Step in Plant Sulfur Assimilation

“…The recently determined crystal structure of an apoenzyme form of the enzyme from the purple sulfur bacterium A. vinosum reveals movement of ␣12 to open the active site (46). Although multiple ATP sulfurylases from different organisms have been crystallized and structurally characterized, functional analysis of these enzymes is limited to the two histidines near the active site that correspond to His-252 and His-255 of GmATPS (63,64). The HXXH motif was originally identified in nucleotidyltransferases and type I amino acyltransferases (65)(66)(67).…”

“…In these enzymes, the conserved histidines are directly involved in cleavage of the ␣/␤-phosphate bond. Mutagenesis of the HXXH motif in the ATP sulfurylase domain of human and mouse PAPS synthetase led to reduced specific activity (63,64), but more detailed steady-state kinetic analysis of the effect on function was not performed. To date, no structure-function analysis of other active site residues of an ATP sulfurylase from any other species has been reported.…”