Title

Structural and functional studies of protein phosphatase-1

Date of Award

1998

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Ernest Y. C. Lee, Committee Chair

Abstract

A mutational analysis of rabbit skeletal muscle protein phosphatase-1 was performed by site-directed mutagenesis of recombinant protein expressed in E. coli. This was based on sequence alignment which showed the existence of a number of invariant residues when eukaryotic Ser/Thr protein phosphatases were compared with bacteriophage phosphatases and adenosine tetraphosphatase. Conserved histidine and aspartate residues which may be involved in metal binding were mutated to test the hypothesis that PP1 is a metalloprotein. The residues which were mutated were H66, H125, H173, H248, D64, D71, D92, D95, N124 and R96. The results showed that mutation of H66, H248, D64 and D92 resulted in severe loss of catalytic function. Mutation of D95, N124 and R96 also led to loss of function, while attempts to mutate H125 and H173 led to production of insoluble, inactive proteins. The results of the mutational analysis are consistent with the recently described crystal structure of PP1, which revealed that PP1 possesses a bimetallic center at the active site. The behavior of the D95, R96 and N124 mutants supports a catalytic mechanism involving nucleophilic attack by a hydroxide ion with H125 functioning as a proton donor to the leaving alcohol group.The effects of addition of divalent metals to the growth media on the expression of recombinant PP1 in E. coli was examined. Our findings show that the presence of Mn$\sp{2+}$ or Co$\sp{2+}$ is required for the expression of recombinant PP1 with high specific activities rather than a requirement for expression of the enzyme. The outcome is that at least three functionally distinct enzymes can be isolated, i.e., rPP1$\rm\sp{-Me}$, rPP1$\rm\sp{Mn}$ and rPP1$\rm\sp{Co}$. Our evidence supports the idea that rPP1$\rm\sp{Mn}$ and rPP1$\rm\sp{Co}$ represent two different metalloprotein forms of PP1; moreover, the effects of these metals on the biosynthesis of the PP1 also indicates that the presence of these metals may be important in the folding process in vivo.The interaction sites of PP1 and inhibitor 2 that are involved in the formation of the ATP/Mg-dependent holoenzyme (PP1/I-2 complex) were investigated by trace-labeling and site-directed mutagenesis. This trace-labeling study demonstrated that the peptide from Ile133 to Arg187 may be involved in the interaction of these two proteins.The yeast two hybrid system was used to screen a human cDNA library for putative PP1 binding proteins. Ten positive clones were identified. One of these, clone 45 was found to be a partial clone of the coding sequence of a known human gene, HCG V, of previously unknown function. (Abstract shortened by UMI.)

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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