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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Deyrup, AT; Krishnan, S; Cockburn, BN; Schwartz, NB

Published Date

  • April 17, 1998

Published In

Volume / Issue

  • 273 / 16

Start / End Page

  • 9450 - 9456

PubMed ID

  • 9545271

Pubmed Central ID

  • 9545271

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.273.16.9450

Language

  • eng

Conference Location

  • United States