Focus on phosphohistidine

Abstract

Phosphohistidine has been identified as an enzymic intermediate in numerous biochemical reactions and plays a functional role in many regulatory pathways. Unlike the phosphoester bond of its cousins (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoramidate (P-N) bond of phosphohistidine has a high DeltaG degrees of hydrolysis and is unstable under acidic conditions. This acid-lability has meant that the study of protein histidine phosphorylation and the associated protein kinases has been slower to progress than other protein phosphorylation studies. Histidine phosphorylation is a crucial component of cell signalling in prokaryotes and lower eukaryotes. It is also now becoming widely reported in mammalian signalling pathways and implicated in certain human disease states. This review covers the chemistry of phosphohistidine in terms of its isomeric forms and chemical derivatives, how they can be synthesized, purified, identified and the relative stabilities of each of these forms. Furthermore, we highlight how this chemistry relates to the role of phosphohistidine in its various biological functions.

Advances in development of new tools for the study of phosphohistidine

“…Unlike τ-pHis, the rate constant for dephosphorylation of π-pHis decreases smoothly between pH 2 and 4, and remains approximately constant over the pH range 4-6; above pH 6 (~pKa of imidazole nitrogen of π-pHis) to pH 9 the rate constant decrease slowly again before a further decrease above pH 9, which is approximately the pKa of the amine (~9.6). [62][63][64] Nonetheless, the data clearly show both π-His (pH 9-11, 46°C) 63 and τ-pHis (pH 8-10, 80.5°C) 62 are stable in solution for extended periods provided the right conditions are used. Hence, application of pHis standards in experiments such as ELISA, chromatography and dot blots with protein conjugates, or any other test where the pHis standard is needed, should be possible.…”

“…The chemistry of pHis amino acid was first studied by Hultquist et al 62,63 and has been covered extensively in a review by Attwood et al 64 In summary, pHis residues contain a weak phosphoramidate bond with the phosphoryl group being most susceptible to hydrolysis when the imidazole nitrogen is protonated (Scheme 1). 62,63 Hence, the rate of hydrolysis of the pHis is dependent on pH.…”

“…Use of the free amino-acid phosphofurylalanine 6 allowed the raising of antibodies, but these only detected the antigen, and not natural pHis. 98 The phosphopyrrole 7 was later synthesized by Attwood et al, 64 but the polyclonal antibodies raised against this epitope detected only the analogue and not pHis. Following Schenkels' proposal of phosphofurylalanine 6 as a potential pHis analogue, Lilley et al 99 synthesized phosphothiophene 8.…”

Acid-Labile Histone Phosphorylations

“…Phosphorylation of these amino acids results in the formation of a P-N (phosphoramidate) bond, which is, unlike the P-O bond, acid-labile and base stable [5]. Although these phosphorylations occur naturally in comparable levels, they have been usually missed since many techniques classically used to study protein phosphorylation involve acidic conditions [6]. Furthermore, it has been relatively more challenging to study the functions of acid labile histone phosphorylations since the phosphate groups are quickly hydrolyzed when peptides containing these modifications are used as immunogens to raise site-specific antibodies, which makes it harder to study their physiological roles.…”

“…Furthermore, it has been relatively more challenging to study the functions of acid labile histone phosphorylations since the phosphate groups are quickly hydrolyzed when peptides containing these modifications are used as immunogens to raise site-specific antibodies, which makes it harder to study their physiological roles. For this reason, new methods to synthesize phosphohistidine and phosphoarginine peptides with improved thermodynamic stability have been developed [6,7]. This review focuses on 2 major types of acid-labile histone phosphorylations, occurring on arginine and histidine.…”