Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases.
The reaction mechanism of protein tyrosine phosphatases (PTPases) and dual-specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site-directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site-directed mutagenesis. The D383N mutant enzyme exhibits a 150-fold reduction in kcat, with Kw only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two-domain structure for Cdc25, with a regulatory N-terminal and a catalytic C-terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three-dimensional model for the core region of Cdc25. By comparing this three-dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.
Eckstein
J W
JW
Mitotix, Inc., Cambridge, Massachusetts 02139, USA.
Beer-Romero
P
P
Berdo
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D015007:Yersinia / Q000201:enzymology; D020687:cdc25 Phosphatases
D018797:Cell Cycle Proteins; D017931:DNA Primers; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases