Question

While the functional involvement of protein tyrosine sulfation has yet to be fully elucidated, it has been shown that tyrosine sulfation causes the alteration of biological activity of proteins, proteolytic processing of bioactive peptides, change in half-life of proteins in circulation, and modulation of extracellular protein–protein interactions. Tyrosylprotein sulfotransferases (TPSTs) catalyze the transfer of a sulphonate moiety from 30 -phosphoadenosine-50 -phosphosulfate (PAPS) to the hydroxyl group of protein-bound tyrosine residue to form a tyrosine O-sulphate ester and 30 -phosphoadenosine-50 -phosphate (PAP).

A picture of active TPST:

b. Assuming the hydroxyl group of tyrosine is not important for activity of TPST and that the aromatic portion of tyrosine is what is required, what mutation could be made in TPST to generate a mutant TPST that could test the hypothesis? Explain your reasoning.

Answer 1

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutantslacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5 to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis.