Small Phosphomonoesters as Probes of Protein-Tyrosine Phosphatase Active Sites
The DSPs and PTPs listed above also were challenged in vitro with free phosphoserine. Each displayed little or no activity towards free phosphoserine. However, the addition of a hydrophobic "handle" to form N-(cyclohexane carboxyl)-O-phospho-L-serine produced a derivative that was hydrolyzed by IphP at rates comparable to that of the avid substrates p-nitrophenyl phosphate and beta-naphthyl phosphate. VHR also hydrolyzed N-(cyclohexane carboxyl)-O-phospho-L-serine, though at a lower rate than IphP. Cdc14 displayed little activity towards N-(cyclohexane carboxyl)-O-phospho-L-serine.
The active site of VHR was mapped and amino acid residues potentially involved in binding N-(cyclohexane carboxyl)-O-phospho-L-serine were identified. The amino acid sequence of VHR was aligned with the amino acid sequences of IphP and Cdc14 to identify the nature of the corresponding residues in IphP and Cdcd14.
Low molecular weight phosphomonoesters have proven to be effective in vitro indicators of protein phosphatase activity. They also have shown potential as diagnostic substrates for specific subclasses of protein phosphatases. However, neither alpha- and beta- naphthyl phosphate nor N-(cyclohexane carboxyl)-O-phospho-L-serine proved to be universal discriminatory substrates for the functional subgroups within the family of protein-tyrosine phosphatases. Indeed, the probability of identifying such a substrate would appear to be relatively low.
Advisor:Peter J. Kennelly; Malcolm Potts; Thomas Keenan
School Location:USA - Virginia
Source Type:Master's Thesis
Keywords:biochemistry and anaerobic microbiology
Date of Publication:09/25/1999