PubMed · 10691360
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D A Taylor. 2000. A less polluting pig.. https://doi.org/10.1289/ehp.108-a14
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Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).
Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P(6)] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P(5), Ins(1,2,3,4,6)P(5), D/L-(1,2,4,5,6)P(5), D/L-(1,2,3,4)P(4), D/L-Ins(1,2,5,6)P(4) and D/L-Ins(1,4,5,6)P(4) in PLP1B mutants as well as the parent variety. In grain extracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D/L-Ins(1,3,4,5)P(4) were observed whereas phytate and the above-mentioned inositol phosphates were present in relatively small amounts. Assignment of D/L-Ins(1,3,4,5)P(4) was corroborated by precise co-chromatography with a commercial Ins(1,3,4,5)P(4) standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and D/L-Ins(1,3,4,5)P(4), which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened further by phytate degradation assays showing that D/L-Ins(1,3,4,5)P(4) did not belong to the spectrum of degradation products generated by endogenous phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P(4) in barley low-phytate mutants are discussed.
In this study, the behavior of enzyme activity as a function of pH and temperature is modeled on the basis of fundamental considerations. A formulation is developed that includes the activation of enzymes with increasing temperatures and the deactivation of enzymes at higher temperature, together with the effect of protonation and hydroxylation on activity at various constant pH levels. The model is calibrated and validated against an extensive set of experimental data on phytases from seven different origins. The percentage variance accounted for (R(2)(adj)), obtained by statistical nonlinear regression analysis on all data sets, was shown to range from 97.6% to 99.5%. The equilibrium constant of protonation and hydroxylation proved to be independent of temperature.