Mutational analysis of the catalytic subunit of muscle protein phosphatase-1.
A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli. The selection of the sites to be mutated was based on sequence alignments which showed the existence of a number of invariant residues when eukaroytic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosinetetraphosphatase [Barton et al. (1995) Eur. J. Biochem. 220, 225-237]. In other studies, it had been shown that PP1 is a metalloprotein [Chu et al. (1996) J. Biol. Chem. 271, 2574-2577], and in this study, we have largely focused on invariant histidine and aspartate residues which may be involved in metal binding. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124, and R96E. The results showed that mutation of H66, H248, D64, and D92 resulted in severe loss of catalytic function. Mutation of D95, N124, and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the involvement of conserved His and Asp residues in metal binding, and are discussed in the context of the recently described crystal structure of PP1 [Goldberg et al. (1995) Nature, 376, 745-753], which reveals that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96, and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group.
Zhang
J
J
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101, USA.
Zhang
Z
Z
Brew
K
K
Lee
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011817:Rabbits
D017931:DNA Primers; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1