ADENOSINE 5'-PHOSPHOSULFATE KINASE FROM PENICILLIUM-CHRYSOGENUM - SITE-DIRECTED MUTAGENESIS AT PUTATIVE PHOSPHORYL-ACCEPTING AND ATP P-LOOPRESIDUES

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 int ermediate acceptor in the transfer of a phosphoryl group from ATP to A PS, Replacement of Ser-107 with alanine yielded an active enzyme with kinetic characteristics similar to those of wild-type APS kinase, Anot her mutant form of the enzyme in which Ser-107 was replaced by cystein e was also active. Covalent modification of Cys-107 eliminated catalyt ic activity, and substrates protected against modification. Mutation o f 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 f ungal APS kinase, but neither it nor any nearby hydroxy amino acid ser ves as an obligatory phophoryl acceptor in the 3'-phosphoadenylylsulfa te synthesis reaction. The results also indicate that the absence of a serine at position 478 in the APS kinase-like C-terminal region of fu ngal ATP sulfurylase does not account for the lack of APS kinase activ ity in that enzyme. However, mutating the ATP P-loop residues in APS k inase to those found in the analogous C-terminal region of fungal ATP sulfurylase eliminated enzyme activity.