3D modeling of the structure of ChimAP.
(A) Schematic representation of the fusion of the first 359 amino acids of the mature human IAP structure to residues 360–430 of the mature human PLAP sequence followed by residues 431–484 of the human mature IAP sequence. (B) 3D representation of the ChimAP structure based on homology to human PLAP, visualized and analyzed using Chimera v1.7 and Swiss-PdbViewer. (C) Top view of the ChimAP structure, indicating the active site serine. Active site metal ions are displayed. The crown domain is represented in red.
Figure 2.
Human IAP and ChimAP sequence alignment and anti-FLAG western blot analysis of FLAG-tagged PLAP, IAP, ChimAP and TNAP.
(A) Protein sequence alignment showing IAP and ChimAP sequence conservation. Substitutions are those introduced by replacement with the PLAP crown domain. (B) Western blot analysis showing that all four enzymes were secreted into the culture medium.
Figure 3.
Time-course of AP activity disappearance upon dissociation of Zn2+ from the active site of IAP, PLAP and ChimAP at pH 7.4 in the presence of 250 µM EDTA.
The slopes between these lines were highly significantly different (p<0.0001).
Figure 4.
Dose-dependent reconstitution of AP activity upon addition of Zn2+ to Chelex-treated alkaline phosphatases at pH 7.4.
Figure 5.
Differences in catalytic behavior for ChimAP at alkaline and physiological pH, analyzed via pNPP substrate saturation curves and corresponding Lineweaver-Burk plots.
Table 1.
Enzyme constants (± SD) using the artificial substrate p-nitrophenylphosphate (pNPP) at alkaline and physiological pH
Figure 6.
Efficiency of IAP, PLAP, ChimAP and TNAP at pH
Plots are representative of three separate experiments.
Figure 7.
Efficiency of IAP, PLAP, ChimAP and TNAP using the physiological substrate PPi, measured as the rate of phosphate formation versus substrate concentration.
Plots are representative of three different experiments.
Figure 8.
Efficiency of IAP, PLAP, ChimAP and TNAP using the pathophysiological substrate LPS, measured as the rate of phosphate formation versus substrate concentration.
Plots are representative of three different experiments.
Figure 9.
Efficiency of IAP, PLAP, ChimAP and TNAP using the physiological substrate PLP, measured as the rate of phosphate formation versus substrate concentration.
Plots are representative of three different experiments.
Table 2.
Kinetic parameters (± SD) of recombinant enzymes with physiological substrates at physiological pH.
Figure 10.
Integrated competition experiments for the enzymes specified between 0.1i and LPS at the indicated concentrations.
Figure 11.
Active site inhibition of FLAG-tagged IAP (A), PLAP (B) and ChimAP (C) activity at pH 9.8 versus pNPP concentration for the indicated concentrations of the uncompetitive inhibitor L-Phe.
The lines represent fitted inhibition curves. Inset: respective double-reciprocal plots of FLAG-tagged PLAP, IAP and ChimAP activity in the presence or absence of the indicated concentrations of L-Phe.
Table 3.
Uncompetitive inhibition with L-amino acids, expressed at IC50 (± SD).
Figure 12.
(A) Plot of residual enzyme activity in the exponential phase of enzyme inactivation for FLAG-tagged PLAP, IAP, ChimAP and TNAP, showing the effect of the denaturing agent guanidinium hydrochloride. (B, C). Lines in B and C are representative of three separate experiments. Heat inactivation of FLAG-tagged enzymes PLAP, IAP, ChimAP and TNAP. The enzymes were incubated for 10 min at different temperatures (25–100°C) (B) or for different time intervals at 65°C (C).
Table 4.
Enzyme stability when exposed to guanidinium chloride (GndCl) or heat.
Figure 13.
Modeling of the predicted effect of the S429E substitution on the active site environment of the superimposed structures of IAP, PLAP and ChimAP.
IAP, PLAP and ChimAP are shown in orange, white and blue, respectively.