Correlation of Heme Binding Affinity and Enzyme Kinetics of Dehaloperoxidase

Abstract

Chemical and thermal denaturation of dehaloperoxidase-hemoglobin (DHP) was investigated to test the relative stability of isoforms DHP A and DHP B and the H55V mutant of DHP A with respect to heme loss. In thermal denaturation experiments, heme loss was observed at temperatures of 54, 46, and 61 °C in DHP A, DHP B, and H55V, respectively. Guanidinium hydrochloride (GdnHCl)- and urea-induced denaturation was observed at respective concentrations of 1.15 ± 0.01 M DHP A and 1.09 ± 0.02 M DHP B, and 5.19 ± 0.05 M DHP A and 4.12 ± 0.14 M DHP B, respectively. The binding affinity of heme appears to be significantly smaller in both isoforms of DHP than in myoglobins. This observation was corroborated by heme transfer experiments, in which heme was observed to transfer for DHP A and B to horse skeletal muscle myoglobin (HSMb). GdnHCl-induced denaturation suggests a threshold of 1 mM for stabilization by binding of the inhibitor 4-bromophenol (4-BP). Concentrations of 4-BP greater than 1 mM caused destabilization. Urea-induced denaturation showed only destabilizing effects from phenolic ligand binding. Heme transfer experiments from DHP to HSMb further support the hypothesis that the binding of halophenols to DHP facilitates the removal of the heme. Thermal denaturation assessed via UV-visible spectroscopy and that assessed by differential scanning calorimetry (DSC) are both in agreement with chemical denaturation experiments and show that the denaturing abilities of the halophenols improve with the size of the para halogen atom in 4-XP, where X = iodo, bromo, chloro, or fluoro (4-IP > 4-BP > 4-CP > 4-FP), and the number of halo substituents as in 2,4,6-tribromophenol (2,4,6-TBP > 4-BP). DHP B, which differs in five amino acids, is less stable than DHP A with ΔHcal and Tm values of 165.1 kJ/mol and 47.5 °C compared to values of 183.3 kJ/mol and 50.4 °C for DHP B and DHP A, respectively. Kinetic studies verified that DHP B has a catalytic efficiency (kcat/Km) ∼5-6 times greater than that of DHP A but showed an increased level of substrate inhibition in DHP B for both 2,4,6-TCP and 2,4,6-TBP. An inverse correlation between protein stability with respect to heme loss and catalytic efficiency is suggested on the basis of the fact that the heme in DHP B has a stability lower than that of DHP A but a catalytic efficiency higher than that of DHP A.

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme

“…3c, d ) at a limiting hydrogen peroxide concentration (100 μM). Compared to the dehaloperoxidase from Amphitrite ornata 29 , an enzyme with a globin evolutionary heritage, C45 improves 5-fold on the natural enzyme’s catalytic efficiency at this limiting H 2 O 2 concentration ( k cat / K m (TCP) = 4.2 × 10 4 M −1 s −1 vs. k cat / K m (TCP) = 8.1 × 10 3 M −1 s −1 for DHP A). We predict k cat and k cat / K m (TCP) values at non-limiting H 2 O 2 concentrations also to be significantly higher than for the natural enzyme.…”

Structure and heme-binding properties of HemQ (chlorite dismutase-like protein) from Listeria monocytogenes

“…The transfer of the prosthetic group from holo-LmCld to excess apo-myoglobin (apo-Mb) was monitored photometrically by the increase of absorbance at 409 nm, similarly as reported previously [20] . Apo-myoglobin (from horse heart, Sigma) was prepared by using a modified extraction method by Teale [21] .…”

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

“…Stability and folding studies show that DHP is much more resistant to precipitation by 2,4,6-TXP. 23 The fluoride titration experiments clearly show that DHP A ( Figure 5)and HRP have different substrate binding profiles. During the titration, the fluoride anion will bind to heme Fe to form the 6-coordinated high-spin (6cHS) heme−fluoride adduct, which is indicated by the charge transfer band CT1 at 605 nm for DHP and 611 nm for HRP, as shown in Figure 6.…”

“…22 The internal phenol binding sites also have structural relevance because they apparently increase the resistance of DHP to denaturation under certain conditions. 23 Because phenols are usually considered denaturants, this property combined with the catalytic rate suggests that DHP plays a unique role as a detoxification enzyme in A. ornata. Thus, continued exploration of the interaction of inhibitor binding and enzyme kinetics provides further evidence that the substrate interactions in DHP are not adventitious, but rather indicative of a true function.…”