Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase

Abstract

The time-resolved kinetics of substrate oxidation and cosubstrate H2O2 reduction by dehaloperoxidase-hemoglobin (DHP) on a seconds-to-minutes time scale was analyzed for peroxidase substrates 2,4,6-tribromophenol (2,4,6-TBP), 2,4,6-trichlorophenol (2,4,6-TCP), and ABTS. Substrates 2,4,6-TBP and 2,4,6-TCP show substrate inhibition at high concentration due to the internal binding at the distal pocket of DHP, whereas ABTS does not show substrate inhibition at any concentration. The data are consistent with an external binding site for the substrates with an internal substrate inhibitor binding site for 2,4,6-TBP and 2,4,6-TCP. We have also compared the kinetic behavior of horseradish peroxidase (HRP) in terms of kcat, Km(AH2) and Km(H2O2) using the same kinetic scheme. Unlike DHP, HRP does not exhibit any measurable substrate inhibition, consistent with substrate binding at the edge of heme near the protein surface at all substrate concentrations. The binding of substrates and their interactions with the heme iron were further compared between DHP and HRP using a competitive fluoride binding experiment, which provides a method for quantitative measurement of internal association constants associated with substrate inhibition. These experiments show the regulatory role of an internal substrate binding site in DHP from both a kinetic and competitive ligand binding perspective. The interaction of DHP with substrates as a result of internal binding actually stabilizes that protein and permits DHP to function under conditions that denature HRP. As a consequence, DHP is a tortoise, a slow but steady enzyme that wins the evolutionary race against the HRP-type of peroxidase, which is a hare, initially rapid, but flawed for this application because of the protein denaturation under the conditions of the experiment.

Heme binding and peroxidase activity of a secreted minicatalase

“…The enzymatic behavior was better described by a hyperbolic dependence with substrate inhibition at high concentrations of the electron donors (Figs B and S3). This behavior has been reported for other enzymes with peroxidatic activity .…”

Conversion of Human Neuroglobin into a Multifunctional Peroxidase by Rational Design

“…The k cat value was also ∼3-fold higher than that of A15C/H64S Ngb and the native enzyme DHP for TCP dehalogenation. Moreover, A15C/H64D Ngb exhibited ∼4.5-fold, ∼16-fold, and ∼42-fold smaller K m values as compared to those of A15C/H64S Ngb, DHP, and the native enzyme, horseradish peroxidase (HRP), respectively . As a result, the overall catalytic efficiencies ( k cat / K m ) were ∼14-fold, ∼51-fold, and ∼3.2-fold higher than those of A15C/H64S Ngb, DHP, and HRP, respectively.…”

Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

“…(3) (for E ref obeying an ordered ternary complex mechanism, such as for SmAA10A 14,18 ) or Eq. (4) (for E ref obeying a ping-pong mechanism, such as for HRP 35 ) (see supplementary "kinetic foundation" for more details).…”

“…We chose HRP as the second H 2 O 2 consuming reference enzyme. HRP is a heme-peroxidase that obeys a ping-pong mechanism 35 . Since SmAA10A has previously been characterized at pH 6.1 and 25°C 14 , the same experimental conditions were used throughout this study.…”

“…The unbound copper was removed by extensive washing with sodium acetate buffer (50 mM, pH 5.0) using an Amicon ultracentrifugation device equipped with a 5 kDa cut-off membrane. The concentrations of Cu-saturated LPMOs were determined by measuring the absorbance at 280 nm using theoretical molar extinction coefficients of 35,200,54,360,46,910, and 75,775 M −1 cm −1 for SmAA10A, TrAA9A, NcAA9C, and ScAA10C, respectively.…”