Fluorescein monophosphates as fluorogenic substrates for protein tyrosine phosphatases.
A series of novel fluorescein monophosphates aimed as substrates for protein tyrosine phosphatases (PTPs) were synthesized and evaluated against fluorescein diphosphate (FDP), the currently used fluorescent substrate for PTPs. In contrast to FDP, which is dephosphorylated to monophosphate and then to fluorescein in a sequential reaction, these monophosphates are dephosphorylated in a single step. This eliminates the complication in assaying PTPs due to the cleavage of the second phosphate group. The kinetic studies of these substrates with PTPs were performed and Michaelis-Menten parameters were obtained. These designed substrates have Km 0.03-0. 35 mM, kcat/Km of 3-100 mM-1 s-1 with CD45 and PTP1B. The results showed that the substrates with negative charge groups on the fluorescein have higher affinities for PTP1B, which are consistent with other observations. In this series, fluorescein monosulfate monophosphate (FMSP) was the best substrate observed. Since FMSP showed large increases in both absorption and fluorescence upon dephosphorylation by PTPs at pH>6.0, it is one of the most sensitive, stable and high affinity substrates reported for PTPs.
Wang
Q
Q
Department of Biochemistry and Molecular Biology, Merck-Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Que. H9R 4P8, Canada. qingping_wang@merck.com
Scheigetz
J
J
Gilbert
M
M
Snider
J
J
Ramachandran
C
C
D005452:Fluoresceins / Q000138:chemical synthesis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000737:chemistry; D063065:Organophosphonates / Q000138:chemical synthesis / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D005452:Fluoresceins; D005456:Fluorescent Dyes; D063065:Organophosphonates; C119004:fluorescein monophosphate; D017493:Leukocyte Common Antigens; D017027:Protein Tyrosine Phosphatases