Investigations into the effect of protein motions and small molecule inhibitors on the PTP YopH

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Alvan Hengge


Catalysis rates of protein tyrosine phosphatases (PTPs) vary by nearly four orders of magnitude despite structurally superimposable active sites and the same catalytic mechanism. The faster PTPs, including YopH, the fastest known, have a flexible WPD-loop containing the catalytic general acid Asp356 that closes over the active site for catalysis. Faster dynamics of this flexible loop are correlated to the faster YopH rate compared to other PTPs. Mutation of the tryptophan 354 residue in the WPD-loop to phenylalanine, tyrosine, or histidine, locks the loop in a quasi-open position. These mutations cause a > 100-fold decrease in rate and loss of general acid catalysis. We report the highest resolution structure of native YopH to date (1.05 ), and structures of the W354Y and W354H mutants between 1.1-1.3 . Crystals of the mutants grown with vanadate show a divanadate glycerol ester in the active site, formed after glycerol was added as a cryoprotectant. Kinetics data show glycerol has no synergistic effect on vanadate inhibition, suggesting the absence of glycerol-vanadate esters as inhibitory species in solution. The electrostatics of the active site and the disabled general acid in these mutants provides a unique environment for the formation of the unusual ester.

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