DELETION AND SITE-DIRECTED MUTAGENESIS OF THE ATP-BINDING MOTIF (P-LOOP) IN THE BIFUNCTIONAL MURINE ATP-SULFURYLASE ADENOSINE 5'-PHOSPHOSULFATE KINASE ENZYME/

The P-loop is a common motif found in ATP- and GTP-binding proteins, T he recently cloned murine ATP-sulfurylase/adenosine 5'-phosphosulfate (APS) kinase contains a P-loop (residues 59-66) in the APS kinase port ion of the bifunctional protein. A series of enzymatic assays covering the multiplicity of functions of this unique protein (reverse ATP-sul furylase, APS kinase, and an overall assay) were used to determine the effect of deleting or altering specific residues constituting this mo tif. In addition to the full-length cDNA construct (1MSK), two deletio n mutants that progressively shortened the N terminus by 34 amino acid s (2MSK) and 70 amino acids (3MSK) were designed to examine the effect s 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 a nd reduced sulfurylase activity. In light of the evident importance of this motif, a number of site-directed mutants were designed to invest igate the contribution of key residues. Mutation of a highly conserved lysine in the P-loop to alanine (K65A) or arginine (K65R) or the foll owing threonine (T66A) to alanine ablated APS kinase activity while le aving ATP-sulfurylase activity intact. Three mutations (G59A, G62A, an d 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-sulfury lase activity without effect on APS kinase activity. A series of highl y conserved flanking cysteines (Cys-53, Cys-77, and Cys-83) were mutat ed to alanine, but none of these mutations showed any effect on either enzyme activity.