Date of Award:

5-2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Committee Chair(s)

Alvan C. Hengge

Committee

Alvan C. Hengge

Committee

Lisa M. Berreau

Committee

Cheng-Wei Tom Chang

Committee

Bradley S. Davidson

Committee

Daren P. Cornforth

Abstract

Phosphorylation of proteins is ubiquitous. Phosphorylation can activate proteins, deactivate proteins, assist in signaling, or serve other roles depending upon the biochemical pathway. Attaching phosphate to proteins is accomplished by enzymes called kinases; removing phosphate from proteins is accomplished by enzymes called phosphatases. Cells must regulate their biochemical pathways, and the antipodal roles of kinases and phosphatases represent the yin-yang of phosphorylation.

Phosphorylation of proteins is known to occur on serine, threonine, and tyrosine. This creates a phosphoester bond. Phosphoester bonds have a phosphorus-oxygen (P-O) bond. The ability of phosphatases to cleave such phosphoester bonds is well studied. Phosphorylation of proteins is also known to occur on lysine, arginine, and histidine. This creates a phosphoramidate bond. Phosphoramidate bonds have a phosphorus-nitrogen (P-N) bond. The ability of phosphatases to cleave such phosphoramidate bonds is not well studied. The research herein involved a side-by-side comparison of the abilities of the known phosphatases to cleave the P-O bond of pNPP and the P-N bonds of N-phenyl phosphoramidate and phosphoryl imidazole.

The enzymes selected were well-studied phosphatases possessing a catalytic motif representative of a class of enzymes. The phosphatases assayed were: the protein tyrosine phosphatase YopH; alkaline phosphatase; the dual-specificity phosphatase VHR; prostatic acid phosphatase, PAcP; PHPT1, the only known phosphohistidine phosphatase; and the serine/threonine phosphatases λPP and PP1. The catalytic efficiencies kcat/KM (s-1M-1), were compared for the respective phosphoramidase and phosphatase activities for each enzyme. Ratios of catalytic efficiencies (kcat/KM)/(kcat/KM) of pNPP over PIm are: YopH – 27; AP – 4.1; VHR – 0.22; PAcP – 1.6; AP – 0.51; and PHPT1 – 0.00007. λPP catalyzed hydrolysis of PIm, although kinetic constants could not be obtained. PP1 exhibited no phosphoramidase activity. The results show that most phosphatase catalytic motifs display catalytic promiscuity by cleaving both phosphoesters and phosphoramidates, but with a pronounced preference for one substrate type versus the other.

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