Paul G. Besant

Focus on Phosphoarginine and Phospholysine

Protein phosphorylation is a common signaling mechanism in both prokaryotic and eukaryotic organisms. Whilst serine, threonine and tyrosine phosphorylation dominate much of the literature there are several other amino acids that are phosphorylated in a variety of organisms. Two of these phosphoamino acids are phosphoarginine and phospholysine. This review will focus on the chemistry and biochemistry of both phosphoarginine and phospholysine. In particular we focus on the biological aspects of phosphoarginine as a means of storing and using metabolic energy (in place of phosphocreatine in invertebrates), the chemistry behind its synthesis and we examine the chemistry behind its highenergy phosphoramidate bond. In addition we will be reporting on the incidence of phosphoarginine in mammalian cells. Similarly we will be reviewing the current findings on the biology and the chemistry of phospholysine and its involvement in a variety of biological systems.

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by (1)H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by (31)P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.

Histidine Phosphorylation in Histones and in other Mammalian Proteins

The investigation of protein histidine phosphorylation has required the development of a number of methods that differ from traditional methods of phosphoprotein analysis that were developed to study phosphorylation of serine, threonine, and tyrosine, which are, unlike phosphohistidine, acid-stable. The investigation of histidine phosphorylation is further complicated by the fact that in mammalian proteins, phosphorylation appears to occur at either 1-N or 3-N positions of the imidazole ring, depending on the source of the kinase. In this review, we describe methods developed for phosphoamino acid analysis to detect phosphohistidine, including the determination of the isoform present, using chromatographic and mass spectrometric analysis of phosphoprotein hydrolysates and 1H- and 31P NMR analysis of intact phosphoproteins and phosphopeptides. We also describe methods for the assay of protein histidine kinase activity, including a quantitative assay of alkali-stable, acid-labile protein phosphorylation, and an in-gel kinase assay applied to histidine kinases. Most of the detailed descriptions of methods are as they are applied in our laboratory to the investigation of histone H4 phosphorylation and histone H4 histidine kinases, but which can be applied to the phosphorylation of any proteins and to any such histidine kinases.

Chapter 14 Protein Histidine Phosphorylation

Publisher Summary This chapter discusses protein histidine phosphorylation, protein histidine kinases (HKs), and protein histidine phosphatases (PHPs) and describes their roles in bacteria, fungi, plants, and mammalian cells. Current methods of detection of phosphohistidine in proteins are also described, including HK assays, phosphoamino acid analysis, and approaches involving mass spectrometric (MS) methods. The HKs in bacteria, fungi, and plants are two-component protein systems composed of two major functional parts: the HK and the response regulator protein. The receptor or sensor protein that has the HK activity exists in the cell membrane as a preformed dimer or in some cases, may dimerize in response to the extracellular signal. One of the simplest ways to confirm that the site of phosphorylation in a phosphoprotein is a particular amino acid is to perform phosphoamino acid analysis. In this process, the protein substrate is phosphorylated using a nucleotide in which the γ-phosphate is radiolabelled, commonly with 32 P, resulting in the formation of a [ 32 P] phosphoprotein product. Phosphohistidine in proteins is directly detected using 31 P and 1 H nuclear magnetic resonance (NMR).