Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs

Abstract

Myeloperoxidase (MPO) catalyzes the breakdown of hydrogen peroxide and the formation of the potent oxidant hypochlorous acid. We present the application of the fluorogenic peroxidase substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in steady-state and transient kinetic studies of MPO function. Using initial kinetic parameters for the MPO system, we characterized under the same conditions a number of gold standards for MPO inhibition, namely 4-amino benzoic acid hydrazide (4-ABAH), isoniazid and NaN3 before expanding our focus to isomers of 4-ABAH and benzoic acid hydrazide analogs. We determined that in the presence of hydrogen peroxide that 4-ABAH and its isomer 2-ABAH are both slow-tight binding inhibitors of MPO requiring at least two steps, whereas NaN3 and isoniazid-based inhibition has a single observable step. We also determined that MPO inhibition by benzoic acid hydrazide and 4-(trifluoromethyl) benzoic acid hydrazide was due to hydrolysis of the ester bond between MPO heavy chain Glu 242 residue and the heme pyrrole A ring, freeing the light chain and heme b fragment from the larger remaining MPO heavy chain. This new mechanism would essentially indicate that the benzoic acid hydrazide analogs impart inhibition through initial ejection of the heme catalytic moiety without prior loss of the active site iron.

Inactivation of myeloperoxidase by benzoic acid hydrazide

“…In order to explore the impact of hydrazide structure on MPO cleavage, we compared whether BAH analogs can cleave MPO native protein, we investigated the effect of BAH analogs on the MPO protein using SDS-PAGE analysis and our aforementioned ability to track the heme activity. Initially, we assessed the extent of cleavage products generated at concentration equal to 5 folds of the approved IC50 values previously determined [17]. These results indicated that BAH was the most potent modification of this cleavage (data not shown).…”

“…We recently reported the inhibition of MPO by BAH and several of its analogs, determining IC 50 values and inhibition constants ( K i ) for each one [17]. A table summarizing these data can be found in Supplementary material (Table.…”

“…In both of these cases, the Fe is lost due to rearrangement of electrons in the architecture of the heme. However, hydrazide-mediated ester bond disruption is a new observation in the field of MPO inactivation [17]. Here we probed the ability of different BAH analogs to fit into the active site channel of MPO and how modifications to the inhibitor might impact their relative access to the HC ester bond position between the pyrrole ring C and a distal HC Glu 242 .…”

“…Analysis of H 2 O 2 /BAH-treated MPO by SDS-PAGE revealed the co-migration of heme with the light chain, suggesting that cleavage of the HC Glu 242 ester and vinyl- HC Met 243 sulfonium preceded loss of the LC Asp 94 ester bond. Indeed, H 2 O 2 /BAH- induced shifts in heme absorption were also consistent with the disruption of its vinyl-sulfonium linkage [17]. The molecular mechanism by which this cleavage takes place and the role of this cleavage in inhibition of MPO remains to be elucidated.…”

“…Recently, we reported that incubation of benzoic acid hydrazide (BAH) with MPO in the presence of H 2 O 2 causes a disruption of the linkages that occurred between the heme b and MPO heterodimer subunits [17]. Analysis of H 2 O 2 /BAH-treated MPO by SDS-PAGE revealed the co-migration of heme with the light chain, suggesting that cleavage of the HC Glu 242 ester and vinyl- HC Met 243 sulfonium preceded loss of the LC Asp 94 ester bond.…”