Investigation of the Cleavage of the Ester Bond in Phosphate Monoesters: Enzymatic and Solution Studies of Phosphoryl Transfer Reactions and Comparison with Analogous Sulfuryl Transfer Reactions.
Abstract
The phosphate ester bond is a ubiquitous species in biochemical systems. Prior studies have elucidated many details of the mechanisms by which this bond is broken. Here this system was further studied through the evaluation of the kinetics of the ester bond cleavage in solution and in enzymatic reactions. The role of solvation was explored by a series of experiments using the extrathermodynamic assumption, which allowed a detailed thermodynamic accounting for the effects of solvent on the reaction. Insights gained into the structure of the transition state through these studies were then applied to the evaluation of several enzymatic systems. The principal enzyme examined was the protein tyrosine phosphatase from Yersinia. Kinetic isotope effects, which provide information on the extent of bond cleavage and charge delocalization in the transition state, were the primary tools of this phase of the study. The mechanism was elucidated through the use of site-directed mutagenesis, which allowed the identification of key roles of particular residues in this enzyme. Further kinetic isotope effect studies were performed with the serine/threonine protein phosphatase-2C, serine/threonine protein phosphatase Lambda, and dual specific vaccinia-Hi-related enzymes for comparison with the Yersinia protein tyrosine phosphatase. The chemistry of sulfation has an important role in biochemical systems, but it is not as ubiquitous as that of phosphoryl transfer. The sulfate ester bond has long been held as a close relative of the phosphate ester bond, and it has been proposed that the mechanism of cleavage is similar in spite of a marked difference in activation parameters. In order to broaden the base of knowledge on these related mechanisms, kinetic and kinetic isotope effect studies were conducted with p-nitrophenyl sulfate to parallel many of those done with the phosphate.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 1999
- Accession Number
- ADA365158
Entities
People
- Richard H. Hoff
Organizations
- Utah State University