Date of Award:


Document Type:


Degree Name:

Doctor of Philosophy (PhD)


Chemistry and Biochemistry

Committee Chair(s)

Alvan C Hengge


Alvan C Hengge


Lisa M. Berreau


Cheng-Wei Tom Chang


Bradley S. Davidson


Marie K. Walsh


"Catalytic promiscuous" enzymes, which possess additional activities besides their "native" activity, albeit with a lower efficiency than the main reaction, have become a new frontier for biochemistry and have received considerable attention. Catalytic promiscuity has been suggested to contribute to enzyme evolution through the mechanism of gene duplication followed by specialization of one of the two copies for the new function. Mimicking this evolutionary shortcut could also provide a more efficient route to changing the function of proteins by directed evolution. The promiscuous phosphatase PP1 is a member of the phosphoprotein phosphatase (PPP) gene family, which is critical for the control of many cellular pathways by antagonizing the effects of protein phosphorylation mediated by kinases. The catalytic promiscuity of PP1&gamma WT and two mutants has been investigated with a set of monoanionic and dianioic phosphester substrates. PP1&gamma is an effective catalyst for the hydrolysis of both monoanionic and dianionic phosphate-ester based substrates 1-5, with second-order rate accelerations that fall within the narrow range of 1011 to 1013. While the transition states of the uncatalyzed hydrolysis reactions of these substrates differ, those for the PP1&gamma-catalyzed reactions are similar. Thus, the enzyme catalyzes the hydrolysis of these substrates by transition states that are controlled by the active site environment more than by the intrinsic nature of the substrates. The reason for the inability of PP1&gamma to catalyze the hydrolysis of a sulfate ester is unclear, and unexpected, since the charge and transition state of this substrate are well within the range of those of the phosphorus-based substrates that are effectively catalyzed. Inhibition experiments suggest that the PP1&gamma active site is tolerant of variations in the geometry of bound ligands. This characteristic may permit the effective catalysis even of substrates whose steric requirements may result in perturbations to the positioning of the transferring group, both in the initial enzyme-substrate complex and in the transition state. The conservative mutation of arginine 221 to lysine results in a mutant that more effectively catalyzes monoanionic substrates than the native enzyme. The surprising result in substrate preference from a single, conservative mutation lends support to the notion that mutations following gene duplication can result in an altered enzyme with different catalytic capabilities and preferences, and may, following subsequent mutations, provide a pathway for the evolution of new enzymes.




This work made publicly available electronically on July 30, 2012.

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