Structure and function studies of mammalian low molecular weight phosphotyrosyl protein phosphatases using NMR spectroscopy

Ming-Ming Zhou, Purdue University

Abstract

A scheme for the purification of recombinant bovine heart and human placental low molecular weight phosphotyrosyl protein phosphatases (BHPTP, HCPTP-A, and -B) was developed. The recombinant enzymes were characterized and found to be kinetically identical to authentic enzymes. Trapping, isolation, and identification of a phosphoenzyme intermediate using $\sp{31}$P NMR spectroscopy provided direct evidence for an enzymatic mechanism in which a cysteine residue is employed as a nucleophile in catalysis by this group of phosphatases. A novel approach using an MLEV-17 sequence was developed to identify histidine C$\sp{\varepsilon 1}$H resonances of proteins. This technique can be readily applied to proteins dissolved directly in D$\sb2$O and eliminates the necessity for an exhaustive exchange of NH to ND. With this technique, histidine peaks of BHPTP, HCPTP-A and -B were readily detected and assigned using corresponding histidine mutants. Analysis of the histidine p$K\sb{\rm a}$'s and examination of $\sp1$H NMR spectra of the proteins indicated that His-66 and His-72 were conserved in these phosphatases, and revealed significant differences in secondary structure between the two human isoenzymes. Titration of the enzymes with vanadate and ketomalonic acid suggested that His-72 and His-66 are likely at substrate binding site of the enzymes, interacting with a phosphate group and peptide moiety of a physiological substrate, respectively. Using a similar approach, a probable active site histidine residue of the 100-kDa human prostatic acid phosphatase was identified using the strong competitive inhibitors, L-(+)-tartrate and the early transition metal oxoanion, tungstate. Furthermore, extensive studies of the histidines of the 45-kDa Escherichia coli acid phosphatase led to the conclusion that two histidines and three arginines are at enzyme active site and that the enzymatic nucleophile of His-17 in RHGXRXP motif appears to possess an unusually low p$K\sb{\rm a}$ (below 2.0) which is likely caused by the close proximity of three arginines, especially Arg-92. Sequence-specific backbone $\sp1$H and $\sp{15}$N resonance assignments and secondary structure studies were conducted using 3D NOESY-HMQC and TOCSY-HMQC NMR experiments on a uniformly $\sp{15}$N-labeled BHPTP in conjunction with several specific $\sp{15}$N-amino acid labeled proteins. These results will be useful for the determination of the solution structure of BHPTP using NMR techniques.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry

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