PNPP

An extra phosphorylation of Na+,K(+)-ATPase by paranitrophenylphosphate (PNPP): evidence for the oligomeric nature of the enzyme

J Biochem 1994 Dec;116(6):1360-9.7706230 10.1093/oxfordjournals.jbchem.a124688

Paranitrophenylphosphate (PNPP) induced fluorescence changes in fluorescence isothiocyanate (FITC)-labeled Na+,K(+)-ATPase preparations. The extents of changes were similar to those induced by acetylphosphate (AcP) accompanying accumulation of a K(+)-sensitive phosphoenzyme (E2P) and an ouabain bound phosphoenzyme in the presence of Mg2+ and 16 mM Na+. Phosphoenzymes formed from [32P]PNPP were shown to turn over. The ratio of the maximum amount of the phosphoenzyme formed from PNPP to that of the phosphoenzyme formed from ATP and that of the ouabain-enzyme complex under steady-state conditions was shown to be close to 1:0.5:1. Such extra phosphorylation has hitherto only been observed in a transient state with the additions of high concentrations of ATP [Peluffo, R.D., Garrahan, P.J., and Rega, A. (1992) J. Biol. Chem. 267, 6596-6601]. Our data are compatible with the simultaneous presence of high and low affinity ATP-binding sites in Na+,K(+)-ATPase [Hamer, E. and Schoner, W. (1993) Eur. J. Biochem. 213, 743-748]. The maximum amount of paranitrophenol-sensitive fraction to synthesize [32P]PNPP in fully accumulated ADP-sensitive phosphoenzyme (E1P) from [32P]ATP was around 1/4 of the amount of ouabain-enzyme complex. These data and others indicate that a much higher degree of oligomerization, rather than (alpha beta)2, may be the functional unit of the enzyme in the membranes.

Kinetic behaviour of calf intestinal alkaline phosphatase with PNPP

Indian J Biochem Biophys 2013 Feb;50(1):64-71.23617076

The hydrolysis of p-nitrophenyl phosphate (PNPP) by calf intestinal alkaline phosphatase (CIAP) was investigated with respect to kinetic parameters such as V(max), K(m) and K(cat) under varying pH, buffers, substrate concentration, temperature and period of incubation. Highest activity was obtained with Tris-HCl at pH 11, while in the case of glycine-NaOH buffer the peak activity was recorded at pH 9.5. The enzyme showed the following kinetic characteristics with PNPP in 50 mM Tris-HCl at pH 11 and 100 mM glycine-NaOH at pH 9.5 at an incubation temperature of 37 degrees C: V(max), 3.12 and 1.6 micromoles min(-1) unit(-1); K(m), 7.6 x 10(-4) M and 4 x 10(-4) M; and K(cat), 82.98 s(-1) and 42.55 s(-1), respectively. CIAP displayed a high temperature optimum of 45 degrees C at pH 11. The kinetic behaviour of the enzyme under different parameters suggested that the enzyme might undergo subtle conformational changes in response to the buffers displaying unique characteristics. Bioprecipitation of Cu2+ from 50 ppm of CuCl2 solution was studied where 64.3% of precipitation was obtained. P(i) generated from CIAP-mediated hydrolysis of PNPP was found to bind with copper and precipitated as copper-phosphate. Thus, CIAP could be used as a test candidate in bioremediation of heavy metals from industrial wastes through generation of metal-phosphate complexes.

[Relationship between the ratio of alkaline phosphatase activity determined with PNPP or PP as substrates and alkaline phosphatase isozymes]

Rinsho Byori 1987 Jul;35(7):779-84.3669390

Solid-Phase Synthesis of Megamolecules

J Am Chem Soc 2020 Mar 11;142(10):4534-4538.32105451 PMC8672447

This paper presents a solid-phase strategy to efficiently assemble multiprotein scaffolds-known as megamolecules-without the need for protecting groups and with precisely defined nanoscale architectures. The megamolecules are assembled through sequential reactions of linkers that present irreversible inhibitors for enzymes and fusion proteins containing the enzyme domains. Here, a fusion protein containing an N-terminal cutinase and a C-terminal SnapTag domain react with an ethyl p-nitrophenyl phosphonate (PNPP) or a chloro-pyrimidine (CP) group, respectively, to give covalent products. By starting with resin beads that are functionalized with benzylguanine, a series of reactions lead to linear, branched, and dendritic structures that are released from the solid support by addition of TEV protease and that have sizes up to approximately 25 nm.

Synthesis of Cyclic Megamolecules

J Am Chem Soc 2018 May 23;140(20):6391-6399.29723476 10.1021/jacs.8b02665

This paper describes the synthesis of giant cyclic molecules having diameters of 10-20 nm. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (PNPP) and benzylguanine (BG) groups, respectively. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. Because these are coupled first- and second-order processes, an analysis of product yields from reactions performed at a range of linker concentrations gives rate constants for cyclization. We determined these to be 9.7 × 10-3 s-1, 2.3 × 10-3 s-1, and 8.1 × 10-4 s-1 for the dimer, tetramer, and hexamer, respectively. This work demonstrates an efficient route to cyclic macromolecules having nanoscale dimensions and provides new scaffolds that can be generated using the megamolecule approach.