| First Author | Deyrup AT | Year | 1998 |
| Journal | J Biol Chem | Volume | 273 |
| Issue | 16 | Pages | 9450-6 |
| PubMed ID | 9545271 | Mgi Jnum | J:47047 |
| Mgi Id | MGI:1202528 | Doi | 10.1074/jbc.273.16.9450 |
| Citation | Deyrup AT, et al. (1998) Deletion and site-directed mutagenesis of the ATP-binding motif (P-loop) in the bifunctional murine ATP-sulfurylase/adenosine 5'-phosphosulfate kinase enzyme. J Biol Chem 273(16):9450-6 |
| abstractText | The P-loop is a common motif found in ATP- and GTP-binding 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 P-loop. 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. |
