Researches On The Enzymatic Hydrolysis Of Pyrophosphate By Human Serum Transferrin And Bovine Lactoferrin

Protein phosphorylation/dephosphorylation, as a regulatory mechanism, plays an important role in signal transduction, gene transcription, DNA replication, cell division, and many other physiological processes. Many biological activitives of important proteins and enzymes are adjusted by reversible phosphorylation.The transferrins, including serum transferrin(TF), lactoferrin(LF),ovotransferrin(OTF), which have the homologous gene, similar functions and structure, are a superfamily of nonheme proteins with a main function to transport Fe3+ and control Fe3+ homeostasis in cells. The 70~80kDa single-chain peptide folds two duplicated lobes, each of which binds one Fe3+ and one carbonate anion (the synerigistic anion) via four residues: two tyrosines, an aspartic acid, and a histidine. It was reported that human milk lactoferrin possess phosphatase and ATPase, and ferric-ion binding protein (the bacterial transferrin) can catalyze the hydrolysis of phosphate ester. Thus, it is important to enucleate the catalytic centre and mechanism of transferrins as a phosphatase, which may have a potential significant in the protein phosphorylation process.Here, the kinetic constants for the reactions of human serum apo-transferrin (apo-hTF) and bovine lactoferrin from milk (LF) with pyrophosphate (PPi) and ATP are reported, and the cleavage mechanism of the pyrophosphate by these proteins is investigated, by using 31P NMR and UV-Vis spectroscopy, ESI-MS, and ICP-AES analysis.A new peak at 3.74ppm, reffering to phosphate (Pi), was observed on the 31P NMR spectra when PPi was incubated with apo-hTF for more than 5h in 10mmol·L-1 Hepes buffer at pH 7.40 and 312K. The intensity of this peak increased while the intensity of the PPi peak (-6.59ppm) decreased as the reaction processed, which indicated that apo-hTF can catalyse the convert of pyrophate into phosphate. The reaction followed second-order kinetics with the rate constants of 3.67×10-4, 1.91×10-4, 1.22×10-4, 8.53×10-5L·mmol-1·h-1 for the molar ratioes [PPi]: [apo-hTF] = 16:1, 27:1, 38:1, 50:1, restively. The initial rate constant was 0.010382mmol·L-1·h-1 (zero-order kinetics), Km was 3.600, and vm was 0.00864 mmol·L-1·h-1.When apo-hTF was titrated with PPi in 10mmol·L-1 Hepes buffer at pH 7.40 and 298K, a negative peak at 245nm and negative bands around 290nm were appeared on the UV-Vis difference spectra. The intensity of these peaks was decreased with the addition of PPi, similar to the reaction between PPi and Tyr, indicating that PPi can bind to Tyr residues, the Fe3+-binding sites in apo-hTF, via H-bond (however, the binding of PPi to His was very weak). The binding was saturated when two molar equivalences of PPi wee present, in accordance with the ICP-AES analysis results, which revealed 3.5 P were present in per mol protein. It can be deduced that the mechanism of the pyrophosphate cleavage by apo-hTF consisted of the following steps: binding of PPi to Tyr, the Fe3+-binding sites, via H-bond, and then hyrolysis of PPi into Pi.Ca2+ can slightly accelerate the cleavage of pyrophosphate by apo-hTF, with the rate constant of 2.31·10-4L·mmol-1·h-1 at pH 7.40, 312K.In constrast, holo-hTf has nearly no effect on the above cleavage process, and apo-hTF cannot catalyse the hydrolysis of ATP either. These could be the reason that the cleft between the binding sites was closed in holo-hTF and ATP was too big to reach to the catalytic centre.Lactoferrin, different to holo-hTF, exhibited the pyrophosphase which can catalyse PPi convert into Pi. The reaction also followed second-order kinetics with the rate constants of 3.45·10-4, 2.42×10-4, 1.36·10-4, 3.30×10-5, and 2.50×10-6L·mmol-1·h-1 for the molar [PPi]: [LF] = 20:1, 40:1, 60:1, 80:1, and 100:1, restively. The initial rate constant was 0.00253mmol·L-1·h-1 (zero-order kinetics), Km was 3.539, and vm was 0.026247mmol·L-1·h-1.From our finding, it could give the cue for the research of the roles played by transferrins in protein phosphorylation that apo-hTF and LF can express the enzymatic function of pyrophosphatase, whose first step is the binding of PPi to the Fe3+-binding sites Tyr via H-bond, followed by the hydrolysis of PPi into Pi.