Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.
The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.
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Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393.
Zhou
M M
MM
Stevis
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Davis
J P
JP
Zhang
Z Y
ZY
Van Etten
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D011993:Recombinant Fusion Proteins; D004247:DNA; D017027:Protein Tyrosine Phosphatases