Role of histidine 42 in ascorbate peroxidase: Kinetic analysis of the H42A and H42E variants

Latesh Lad, Martin Mewies, Jaswir Basran, Nigel S. Scrutton, Emma L. Raven

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    To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, μ = 0.10 M, 25.0°C) revealed wavelength maxima [λmax (nm): 397, 509, ≈ 540sh, 644 (H42A); 404, 516, ≈ 538sh, 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 ± 0.3 U·mg-1) was ≈ 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [λmax (nm) = 403, 534, 575sh, 645] and H42E [λmax (nm) = 404, 530, 573sh, 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (Kd) followed by conversion of this intermediate into Compound I (k′1). Values for k′1 and Kd were, respectively, 4.3 ± 0.2 s-1 and 30 ± 2.0 mM (H42A) and 28 ± 1.0 s-1 and 0.09 ± 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H2O2] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH- dependent [pKa = 4.9 ± 0.1 (rAPX) and pKa = 6.7 ± 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pKa for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.
    Original languageEnglish
    Pages (from-to)3182-3192
    Number of pages10
    JournalEuropean Journal of Biochemistry
    Issue number13
    Publication statusPublished - 2002


    • Ascorbate peroxidase
    • Compound I
    • Histidine 42


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