Structural and mechanistic studies of low molecular weight protein tyrosine phosphatases

Shuishu Wang, Purdue University

Abstract

This research examines structure and function relationships of several low molecular weight protein tyrosine phosphatases (low Mr PTPases), which are members of a subgroup of PTPases that is widely if not ubiquitously expressed among living organisms. The low Mr PTPase from Saccharomyces cerevisiae, LTP1, was overexpressed and purified from E. coli. LTP1 has substrate specificity similar to its mammalian homologues. However, it exhibits lower kcat and Km values toward artificial substrates and is subject to relatively strong competitive inhibition by the buffer molecule HEPES. The crystal structure of LTP1 complexed with HEPES showed that there are more aromatic residues on the walls of active site cavity of LTP1 than the mammalian homologous enzymes. These aromatic residues interact favorably with HEPES, and are likely to play an important role in the binding and recognition of natural substrates. The structure of an inactive mutant (LTP1-C13A) complexed with substrate p-nitrophenyl phosphate at a resolution of 1.7 Å confirmed the importance of the aromatic residues on the walls of active site cavity in substrate binding. Moreover, these two structures together confirmed the important role of the rigid structure of the phosphate binding loop in stabilizing the deep binding position of the phosphate group of substrates, thereby facilitating formation of the phosphoenzyme intermediate. The crystal structure of LTP1-C13A complexed with the activator adenine explained a long observed phenomenon that adenine activates the low Mr PTPases. It showed that adenine activates the enzyme by binding in the active site cavity, where together with the side chain of the proton donor aspartic acid it holds a water molecule at a suitable position for a nucleophilic attack on the covalent phosphoenzyme intermediate. A structure of the D129A mutant of a homologous bovine enzyme, in which the proton donor aspartic acid was replaced by alanine, was also determined. The implications of this structure with respect to function of substrate-trapping mutants are discussed.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biochemistry|Molecular biology

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