Deletion and site-directed mutagenesis of the ATP-binding motif (P-loop) in the bifunctional murine ATP-sulfurylase/adenosine 5'-phosphosulfate kinase enzyme.

The Journal of Biological Chemistry
A DeyrupN B Schwartz

Abstract

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 acti...Continue Reading

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