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Adenosine 5 '-phosphosulfate kinase from Penicillium chrysogenum - site-directed mutagenesis at putative phosphoryl-accepting and ATP P-loop residues

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Overview

authors

  • MacRae, Ian
  • Rose, A. B.
  • Segel, I. H.

publication date

  • October 1998

journal

  • Journal of Biological Chemistry  Journal

abstract

  • 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.

subject areas

  • Adenosine Triphosphate
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • DNA Primers
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Organophosphorus Compounds
  • Penicillium chrysogenum
  • Phosphotransferases (Alcohol Group Acceptor)
  • Recombinant Proteins
  • Sequence Homology, Amino Acid
  • Sulfate Adenylyltransferase
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.273.44.28583

PubMed ID

  • 9786849
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Additional Document Info

start page

  • 28583

end page

  • 28589

volume

  • 273

issue

  • 44

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