Title page for ETD etd-092499-034515

Type of Document Master's Thesis
Author Shelton, Thomas Earl
Author's Email Address tshelton@vt.edu
URN etd-092499-034515
Title Small Phosphomonoesters as Probes of Protein-Tyrosine Phosphatase Active Sites
Degree Master of Science
Department Biochemistry and Anaerobic Microbiology
Advisory Committee
Advisor Name Title
Kennelly, Peter J. Committee Chair
Keenan, Thomas W. Committee Member
Potts, Malcolm Committee Member
  • Protein-Tyrosine Phosphatase
  • N-Benzoyl-O-Phospho-L-Serine
  • Cyanobacteria
  • Naphthyl Phosphate
Date of Defense 1999-09-17
Availability restricted
I evaluated the potential of isomers of the low molecular weight phosphomonoester naphthyl phosphate as general diagnostic substrates for differentiating between two families of protein phosphatases: the protein-tyrosine phosphatases [PTPs] and the dual-specificity protein phosphatases [DSPs]. Three PTPs, PTP-1B, Tc-PTPa, and PTP-H1, and three DSPs, Cdc-14, VHR, and IphP, were challenged in vitro with alpha-naphthyl phosphate and beta-naphthyl phosphate. Both the DSPs and PTPs readily hydrolyzed beta-naphthyl phosphate. As expected, the DSPs also hydrolyzed alpha-naphthyl phosphate at rates comparable to beta-naphthyl phosphate and two of the PTPs, PTP-1B and Tc-PTPa, hydrolyzed alpha-naphthyl phosphate at a rate one-tenth that of beta-naphthyl phosphate. However, PTP-H1 hydrolyzed both alpha- and beta- naphthyl phosphate at nearly equal rates. Intriguingly, when challenged with radiolabeled phosphoproteins, PTP-H1 was markedly less stringent, by a factor of 40- to 200- fold, than PTP-1B or Tc-PTPa in its selectivity for [32P]phosphotyrosyl- over [32P]phosphoseryl- proteins in vitro.

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.

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