The Phosphoramidase Competency of Prototypical Phosphatase Catalytic Motifs

The discovery that phosphorylation of proteins occurs on nitrogen by particular kinases raises the question of whether a separate class of phosphoramidases also exists, or if known phosphatases carry out the hydrolysis of phosphoramidates. The phosphoramidase activity of a number of phosphatases with different catalytic motifs was studied using the substrates N-phenylphosphoramidate (N-phPAM) and phosphoryl imidazole (PIm). The phosphatases assayed were: the protein tyrosine phosphatase YopH; alkaline phosphatase; the dual-specificity phosphatase VHR; prostatic acid phosphatase, PAcP; PHPT1, the only known phosphohistidine phosphatase; and, the serine/threonine phosphatases Lambda PP and PP1. The catalytic efficiencies, kcat/KM (s-1M-1), were compared for the respective phosphoramidase and phosphatase activities for each enzyme. Ratios of catalytic efficiencies (kcat/KM)/(kcat/KM) of pNPP over PIm are: YopH - 27; AP - 4.1; VHR - 0.22; PAcP - 1.6; AP - 0.51; and PHPT1 - 0.00007. Lambda PP catalyzed hydrolysis of PIm, although kinetic constants could not be obtained. PP1 exhibited no phosphoramidase activity. The results show that most phosphatase catalytic motifs display catalytic promiscuity by cleaving both phosphoesters and phosphoramidates, but with a pronounced preference for one substrate type versus the other

Effect of osmolytes on regulating the activities of the SSK1 response regulator from Saccharomyces cerevisiae

The multi-step His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells to adapt to osmotic, oxidative and other environmental stresses. The pathway consists of a hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1 and two response regulator proteins, SSK1 and SKN7. Under non-osmotic stress conditions, the SLN1 sensor kinase is active and phosphoryl groups are shuttled through YPD1 to SSK1, therefore maintaining the response regulator protein in a constitutively phosphorylated state. The cellular response to hyperosmotic stress involves rapid efflux of water and changes in intracellular ion and osmolyte concentration. To address the effect of osmolytes on the regulation of this signaling pathway, the individual and combined effects of NaCl and glycerol on phosphotransfer rates within the SLN1-YPD1-SSK1 phosphorelay were examined. In addition, the effect of osmolyte concentration on the half-life of the phosphorylated SSK1 receiver domain in the presence/absence of YPD1 was evaluated. The results show that the combined effects of glycerol and NaCl on the phosphotransfer reaction rates are different from the individual effects of glycerol and NaCl. The combinatory effect is likely more representative of the in vivo changes that occur during hyperosmotic stress. The results revealed that increasing osmolyte concentrations negatively affects the YPD1*SSK1~P interaction thereby facilitating dephosphorylation of SSK1 and activating the HOG1 MAP kinase cascade. At high osmolyte concentrations, the kinetics of the phosphorelay favors production of SSK1~P and inhibition of the HOG1 pathway.A similar multi-step signaling pathway is also utilized by Candida albicans, which is known for adaptation to oxidative stress, morphogenesis, cell wall biosynthesis and virulence in this opportunistic pathogenic yeast. To biochemically characterize major components of this pathway, studies were focused on in vitro reconstitution of the multi-step phosphorelay from C. albicans and biochemical characterization of the CaYPD1 (HPt protein) and CaSSK1 (response regulator protein). The heterologous phosphoryl transfer system SLN1-HK-RR → CaYPD1→ CaSSK1 (or SSK1) was established and examined. The CaYPD1 histidine phosphotransfer protein exhibited similar phosphotransfer specificity in vitro towards the response regulator domain of CaSSK1-RR and SSK1-RR. The half-life of the phosphorylated regulatory domain of CaSSK1-RR was also measured and was approximately 9 min with a corresponding rate constant of 0.078 min-1. This result demonstrates a similar rate of CaSSK1-RR dephosphorylation compared to SSK1-RR suggesting possible functional similarities between these two response regulator proteins.Mutational analysis of the CaSSK1 response regulator domain was also performed. Mutants were expressed, purified and their activity was analyzed using an in vitro phosphorylation assay. Little or no phosphorylation was observed for the CaSSK1-RR D556N mutant. The radiolabel primarily resided with the CaYPD1 protein and did not get transferred to the D556N mutant. Likewise, the D513K mutant was also severely impaired in its ability to be phosphorylated by CaYPD1. The receiver domains of the D556N and the D513K mutants could not be appreciably phosphorylated in vitro indicating that constitutive activation of HOG1 occurs in vivo due to the inability of CaSSK1 to be phosphorylated

Regulation of the oxidation of reduced nicotinamide-adenine dinucleotide in isolated plant mitochondria

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I. Comparison of Translesion Bypass of Guanine–N2 Monoadducts of Mitomycin C and Guanine-N7 Monoadducts of 2,7-diaminomitosene by T7 exo-, Klenow exo-, eta and Klenow exo+ DNA Polymerases. II. Structure-based Design, Synthesis, Structure-conformation and Structure-activity Relationships Studies of D-Phe-Pro-D-Arg-P1’-CONH2 Tetrapeptides with Inhibitory Activity for Thrombin.

The guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2\u27-deoxyguanosine (2,7-DAM-dG) and is noncytotoxic. In part I of this study we tested a potential correlation between the lack of cytotoxicity of 2,7-DAM and the relative ease of bypass of this adduct as compared with the MC adduct. 24-mer and 27-mer templates, adducted at a single guanine either with MC or 2,7-DAM were synthesized and submitted to extension of primers by T7 exo-, Klenow exo-, Klenow exo+, and eta DNA polymerases. The G-N7-2,7-DAM adduct was bypassed by all four polymerases, resulting in the production of a fully extended primer. In sharp contrast, the G-N2-MC monoadduct was not bypassed beyond the adduct position under the same conditions by any of the four polymerases. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5\u2732P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. These results showed for the first time that the dG-N7-2, 7-DAM lesion is non-mutagenic in cell-free systems. In part II of this research structure-based design and molecular docking were employed to design in silico libraries of peptides as potential reversible inhibitors of thrombin. The candidate inhibitors were selected from two original classes of amino acids sequences (1)-D-Phe-Pro-Arg (P1)-D-Pro(P1\u27)-P2\u27-P3\u27-CONH2 and (2)-D-Phe-Pro-D-Arg(P1)-P1\u27-P2\u27-P3\u27-CONH2. For the first time in the field of peptides inhibitors for thrombin we showed that the presence of D-Pro at P1\u27 Position and the use of D-Arg instead of L-Arg at P1 Position is responsible for inhibiting hydrolysis of these of peptides by thrombin, causing these sequences to be inhibitors. In vitro kinetics of thrombin inhibition showed a specific structure-activity relationship at P1\u27 position in the peptide sequence space (2)-D-Phe-Pro-D-Arg(P1)-P1\u27-CONH2. The lead peptides (D-Phe-Pro-D-Arg-D-Ala-CONH2, D-Phe-Pro-D-Arg-D-Thr-CONH2, D-Phe-Pro-D-Arg-D-Cys-CONH2, D-Phe-Pro-D-Arg-D-Ser-CONH2) had competitive or mixed inhibition with respect to thrombin and are characterized by inhibitory constant in the 20-0.8 micromolar range