Identification and pK_a Determination of the Histidine Residues of Human Low-Molecular-Weight Phosphotyrosyl Protein Phosphatases: A Convenient Approach Using an MLEV-17 Spectral Editing Scheme

A useful approach using an MLEV-17 pulse sequence was developed to identify histidine C^ϵ1H magnetic resonances of proteins. This technique can be readily applied to proteins dissolved directly in deuterium oxide solution and eliminates the necessity for an exhaustive exchange of NH to ND. Because of its sensitivity, this technique makes it possible to significantly extend the limitations on protein size. The utility of this spin-lock sequence is demonstrated using ribonuclease, subtilisin, and human prostatic acid phosphatase, with molecular weights ranging from 12K to 100K. With this technique, all three or four of the histidine ¹H NMR signals of two human low-molecular-weight phosphotyrosyl protein phosphatases (HCPTP-A or -B, respectively) were readily detected. Histidine peak assignments were accomplished through the use of histidine to alanine mutants of HCPTP-A and -B and a homologous bovine enzyme. Analysis of the pH titration curves of these signals provided microscopic pK_a's for the histidines in the human enzymes. A comparison of corresponding histidine pK_a values of the two isoenzymes, together with an examination of the ¹H NMR spectra of the proteins, provided evidence of significant differences in secondary structure. Titration of HCPTP-A and -B with vanadate, a strongly bound competitive inhibitor, caused the His-72 peak to appear as two signals at nearly equimolar concentrations of protein and vanadate, while the other histidine peaks were not affected. This is interpreted to mean that His-72 is at the enzyme active site.