Deletion and Site-directed Mutagenesis of the ATP-binding Motif (P-loop) in the Bifunctional Murine Atp-Sulfurylase/Adenosine 5′-Phosphosulfate Kinase Enzyme

Abstract

The P-loop is a common motif found in ATP-and GTPbinding proteins. The recently cloned murine ATP-sulfurylase/adenosine 5-phosphosulfate (APS) kinase contains a P-loop (residues 59 -66) in the APS kinase portion of the bifunctional protein. A series of enzymatic assays covering the multiplicity of functions of this unique protein (reverse ATP-sulfurylase, APS kinase, and an overall assay) were used to determine the effect of deleting or altering specific residues constituting this motif. In addition to the full-length cDNA construct (1MSK), two deletion mutants that progressively shortened the N terminus by 34 amino acids (2MSK) and 70 amino acids (3MSK) were designed to examine the effects of translation initiation before (2MSK) and after (3MSK) the Ploop. The 2MSK protein possessed sulfurylase and kinase activity equivalent to the full-length construct, but 3MSK exhibited no kinase activity and reduced sulfurylase activity. In light of the evident importance of this motif, a number of site-directed mutants were designed to investigate the contribution of key residues. Mutation of a highly conserved lysine in the P-loop to alanine (K65A) or arginine (K65R) or the following threonine (T66A) to alanine ablated APS kinase activity while leaving ATP-sulfurylase activity intact. Three mutations (G59A, G62A, and G64A) addressed the role of the conserved glycines as follows: G64A showed diminished APS kinase activity only, whereas G62A had no effect on either activity. G59A caused a significant decrease in ATP-sulfurylase activity without effect on APS kinase activity. A series of highly conserved flanking cysteines (Cys-53, Cys-77, and Cys-83) were mutated to alanine, but none of these mutations showed any effect on either enzyme activity.

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

“…Since PAPS synthetase is a bifunctional enzyme with at least two reactive centers and multiple binding sites, several factors could lead to loss of overall activity, including loss of kinase activity, loss of sulfurylase activity, or inefficient transfer of APS from sulfurylase to kinase. Furthermore, the possibility that a mutation in one functional portion of the protein can affect the other reaction, as we previously found for a P-loop mutation present in the kinase portion, which resulted in loss of sulfurylase activity (17), complicates interpretation. Thus, in order to more definitively assess the functionality of these motifs in the sulfurylase reaction, individual sulfurylase and kinase assays, as well as overall assays, were performed.…”

“…The PP-loop was found to be present in monofunctional ATP sulfurylases, as well as Escherichia coli NtrL protein and Bacillus subtilis OutB protein (23). The PP-loop is often found in association with other functional motifs/domains, like the P-loop motif (present in the kinase portion) in PAPS synthetases (17) or an amidotransferase domain, a citrulline-aspartate ligase domain, and a nitrilase/amidase domain (16). The other motif, HXXH, is highly conserved among the monofunctional ATP sulfurylases and bifunctional PAPS synthetases, and through a sequence comparison with TagDrelated nucleotidyltransferases, it is hypothesized that this motif is participating in the ␣-␤ phosphodiesterase activity (21).…”

“…Many of these conserved residues are present in motifs that have been implicated in ATP binding (P-loop) (13), phosphoryl transfer (FISP) (14), phosphodiester-cleavage (15), and pyrophosphate binding (PPloop) (16). Recently, we reported on the generation of several site-directed mutants in the ATP-binding motif of the kinase domain (17), as well as the expression of truncated, monofunctional and rearranged domains of the bifunctional enzyme (18). Continuing our elucidation of the important binding motifs and functional residues in the bifunctional enzyme, we have extended our analysis to the sulfurylase portion of the enzyme, where enzyme inactivation via chemical modification, in conjunction with sequence alignment, aided in identifying potentially important arginine and histidine residues (19).…”

Endostatin Has ATPase Activity, Which Mediates Its Antiangiogenic and Antitumor Activities

“…The flexible Walker A motif contains an invariant Lys residue. Mutation of this residue frequently leads to decreased nucleotide-binding capacity (38), therefore, mutants K96A and K96R were generated (Fig. 3A).…”

Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis inCaenorhabditis elegans

“…On the basis of this observed similarity with ABC transporters, it was suggested LIC may be an ATPase (Gill et al, 1994;Hughes et al, 1995); however, no experimental evidence has been published in support of this suggestion. Numerous lines of evidence suggest that a conserved lysine residue within the P-loop of ATPases and other proteins such as kinases, which also contain P-loops, is essential for the role of this domain (Azzaria et al, 1989;Stephens et al, 1995;Deyrup et al, 1998;Wu and Horvitz, 1998). An example is the C. elegans proapoptotic ABC transporter-like gene ced-7.…”