Structural basis of the inhibition of low molecular weight protein-tyrosine-phosphatases

Ming Zhou, Purdue University

Abstract

Pyridoxal 5′-phosphate (PLP) is a strong competitive inhibitor of low Mr protein tyrosine phosphatases. The structural basis of this tight binding of PLP was examined by a variety of methods. Binding constants of a number of PLP analogs were measured with wild-type bovine low Mr protein tyrosine phosphatase (BPTP), and PLP binding constants of some site-specific mutants of BPTP were determined at pH 5.0 through the use of several independent methods. The association of PLP (Ki = 7.6 μM) causes a downfield shift of the His-72 C∈1H resonance in the 1H NMR spectrum of the protein, consistent with a structural alteration in the phosphate-binding loop transmitted through a complex hydrogen bond network that exists between His-72 and Asn-15, which is a residue in the phosphate binding loop. 1H NMR was used to monitor the aldehyde resonance of PLP during titration of a catalytically inactive C12A mutant of BPTP. The aldehydic proton resonance of PLP shifted from 10.43 to 10.26 ppm upon complex formation with the C12A mutant. This resonance occurs far from the region where a hemithioacetal hydrogen would be expected to appear, consistent with the conclusion that the Cys-17 side chain of BPTP does not add to the aldehyde group of PLP. The binding constant of PLP to a C17A mutant was measured and found to be very similar to that exhibited with wild-type protein. These results show that Cys-17 makes virtually no contribution to the tight binding of PLP by BPTP, in contrast to a published report that it is “essential” for binding PLP. On the other hand, Asp-129 of BPTP was found to be very important for binding PLP. The interactions described here should be useful in the design of specific inhibitors of this and related phosphotyrosyl protein phosphatases. The early transition metal oxoanions, especially vanadate, are potent inhibitors of phosphatases. Vanadate inhibits BPTP via a transition state analog geometry. 1H NMR suggests that there is a structural alternation of the phosphate binding loop or associated hydrogen bonding residues upon vanadate binding.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry|Pharmacology

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