Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.
Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.
Aitken
S M
SM
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada.
Turnbull
J L
JL
Percival
M D
MD
English
A M
AM
D001549:Benzamides / Q000737:chemistry; D001665:Binding Sites; D002021:Buffers; D002151:Calorimetry; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000737:chemistry*; D006898:Hydroxylamines / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry; D007538:Isoniazid / Q000737:chemistry; D008956:Models, Chemical; D009539:Nicotinic Acids / Q000737:chemistry; D012457:Salicylamides / Q000737:chemistry; D013379:Substrate Specificity; D013816:Thermodynamics; D017186:Titrimetry
D001549:Benzamides; D002021:Buffers; D006834:Hydrazines; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D007527:Isoenzymes; D009539:Nicotinic Acids; D012457:Salicylamides; C020423:N,N-naphthaloylhydroxylamine; D006418:Heme; C017064:benzyloxyamine; C005703:salicylhydroxamic acid; C028571:nicotinic acid hydrazide; D006735:Horseradish Peroxidase; C038530:N-methylbenzamide; C006696:benzohydroxamic acid; D007538:Isoniazid