H NMR investigation of the influence of interacting sites on the dynamics and thermodynamics of substrate and ligand binding to horseradish peroxidase.
The influence of substrate benzhydroxamic acid (BHA) and iron ligand (cyanide) on the thermodynamics and dynamics of each of the two binding sites of horseradish peroxidase (HRP) isozyme C has been investigated by 1H NMR spectroscopy. A combination of line-width analysis and saturation transfer spectroscopy has allowed the direct determination of the off-rate of substrate and ligand in the absence or presence of the other. These off-rates, together with available dissociation constants obtained by optical spectroscopy (Schonbaum, 1973), provide estimates for kon. The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the 1H NMR determined dynamic and thermodynamic data agree well with those values available in the literature. BHA binding leads to a 200-fold decrease in CN- affinity that arises from a factor greater than 10 decrease in koff(CN-) and greater than 2 x 10(3) decrease in kon(CN-). While a portion of the decrease in kon(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in kon(CN-) and that in koff(CN-) indicates that BHA in the binding pocket blocks the CN- ligation channel and serves as a "gate" to CN- exchange. This view is supported by observing a factor greater than 4 decrease in distal His labile proton exchange with bulk water in HRP-CN upon BHA binding. The ternary complex BHA.HRP-CN is shown to be heterogeneous. While the thermodynamics of BHA and CN- binding appear similar in the two ternary complexes, the BHA on- and off-rates for the two complexes differ by a factor of approximately 10. The two heterogeneous forms interconvert at 25 degrees C at approximately 2 x 10(2) s-1, precluding the determination of any difference in the CN- binding rates by saturation transfer. The greater lability of one of the two ternary complexes is attributed to an alternate orientation of some distal residue that blocks the substrate binding channel in one of the forms. Transferred nuclear Overhauser effects from the heme to BHA in the ternary complex reveal that the BHA substrate is in contact not only with the heme pyrrole D substituents but also with the distal His 42, indicating that the polar side chain of BHA extends well into the distal heme pocket.(ABSTRACT TRUNCATED AT 400 WORDS)
La Mar
G N
GN
Department of Chemistry and Nuclear Magnetic Resonance Facility, University of California, Davis 95616.
Hernández
G
G
de Ropp
J S
JS
D000595:Amino Acid Sequence; D001665:Binding Sites; D003486:Cyanides / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D011487:Protein Conformation; D013816:Thermodynamics
D003486:Cyanides; D006877:Hydroxamic Acids; D008024:Ligands; D006859:Hydrogen; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid