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Biochemistry. 1998 Apr 21;37(16):5383-93.

Electrostatic evaluation of the signature motif (H/V)CX5R(S/T) in protein-tyrosine phosphatases.

Author information

1
MedChem Research IV, Novo-Nordisk A/S, Mâlov, Denmark.

Abstract

The catalytic activity of protein-tyrosine phosphatases (PTPs) is mediated by a cysteine side chain which carries out a nucleophilic attack initiating the phosphate cleavage. Experimentally, it has been observed that the active site cysteine has a remarkably low pKa. In the present study, we have investigated the origin of the low pKa by analyzing the electrostatic properties of four different protein-tyrosine phosphatases: Yersinia PTP (bacteria), PTP1B (human), VHR (human), and low molecular weight phosphatase (bovine). These phosphatases have very low sequence homology and show very low structural similarity. However, they share a common active site motif [the (H/V)CX5R(S/T) sequence] which adopts a unique loop structure. We have applied the so-called single site titration method, which is based on the Poisson-Boltzmann methodology, to (i) study the influence of the architecture of the (H/V)CX5R(S/T) loop on the pKa of the active cysteine and (ii) examine which parts of the active site region stabilize the ionized form of the cysteine. Our results indicate that the architecture of the (H/V)CX5R(S/T) loop has a major impact on the low pKa of the active cysteines. The orientation of the microdipoles generated by the partial charges of the backbone atoms (i.e., the CONHCalpha atoms) is essential for maintaining the low pKa. Further, the electrostatic field generated by these microdipoles has a larger impact than the electrostatic dipole generated by the central alpha-helix. Interactions of the active cysteine with other ionizable side chains play a minor role in stabilizing the thiolate anion. The only ionizable side chain significantly influencing the pKa of the active site cysteine is the arginine, which is an important part of the consensus sequence.

PMID:
9548920
DOI:
10.1021/bi971187i
[Indexed for MEDLINE]

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