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. 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.
Deyrup
A T
AT
Department of Pediatrics, The University of Chicago, Chicago, Illinois 60637, USA.
Krishnan
S
S
Cockburn
B N
BN
Schwartz
N B
NB
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D007700:Kinetics; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013430:Sulfate Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism*
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase