Mutagenesis of the catalytic subunit of rabbit muscle protein phosphatase-1.
We have generated site-directed mutants of the catalytic subunit of rabbit muscle ppase-1. Since it is known that ppase-1 and ppase-2A are highly susceptible to inactivation by sulfhydryl reagents, we have mutagenized the six cysteine residues conserved between these two enzymes to serines. The six mutants were purified to near homogeneity by affinity chromatography on inhibitor-2-Sepharose and characterized. All six exhibited enzymatic activity. These results indicate that the catalytic mechanism of ppase-1 is different from that of the protein tyrosine phosphatases which involve a cysteinyl phosphate intermediate.
Zhang
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Z
Department of Biochemistry & Molecular Biology, University of Miami School of Medicine, FL 33101.
Zhao
S
S
Deans-Zirattu
S
S
Bai
G
G
Lee
E Y
EY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D017931:DNA Primers; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1