Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. site-directed mutagenesis at putative phosphoryl-accepting and ATP P-loop residues.
The properties of Penicillium chrysogenum adenosine 5'-phosphosulfate (APS) kinase mutated at Ser-107 were examined. Ser-107 is analogous to a serine of the E. coli enzyme that has been shown to serve as an intermediate acceptor in the transfer of a phosphoryl group from ATP to APS. Replacement of Ser-107 with alanine yielded an active enzyme with kinetic characteristics similar to those of wild-type APS kinase. Another mutant form of the enzyme in which Ser-107 was replaced by cysteine was also active. Covalent modification of Cys-107 eliminated catalytic activity, and substrates protected against modification. Mutation of Ser-97, of Ser-99, of Thr-103, of Ser-104 to alanine, or of Tyr-109 to phenylalanine also yielded an active enzyme. The cumulative results indicate that Ser-107 may reside in the substrate binding pocket of fungal APS kinase, but neither it nor any nearby hydroxy amino acid serves as an obligatory phophoryl acceptor in the 3'-phosphoadenylylsulfate synthesis reaction. The results also indicate that the absence of a serine at position 478 in the APS kinase-like C-terminal region of fungal ATP sulfurylase does not account for the lack of APS kinase activity in that enzyme. However, mutating the ATP P-loop residues in APS kinase to those found in the analogous C-terminal region of fungal ATP sulfurylase eliminated enzyme activity.
MacRae
I J
IJ
Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
Rose
A B
AB
Segel
I H
IH
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000737:chemistry; D010408:Penicillium chrysogenum / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013430:Sulfate Adenylyltransferase / Q000378:metabolism
D017931:DNA Primers; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D013430:Sulfate Adenylyltransferase