La Mar G N, Hernández G, de Ropp J S
Department of Chemistry and Nuclear Magnetic Resonance Facility, University of California, Davis 95616.
Biochemistry. 1992 Sep 29;31(38):9158-68. doi: 10.1021/bi00153a007.
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)
通过核磁共振氢谱（1H NMR）研究了底物苯甲羟肟酸（BHA）和铁配体（氰化物）对辣根过氧化物酶（HRP）同工酶C两个结合位点的热力学和动力学的影响。结合线宽分析和饱和转移光谱法，可直接测定在不存在或存在另一种物质的情况下底物和配体的解离速率。这些解离速率，连同通过光谱学获得的可用解离常数（Schonbaum，1973），可用于估算结合速率常数（kon）。还通过核磁共振直接测定了氰化物与BHA.HRP复合物结合的解离常数。在所有情况下，1H NMR测定的动力学和热力学数据与文献中的值吻合良好。BHA的结合导致CN-亲和力降低200倍，这是由于koff(CN-)降低了10倍以上，kon(CN-)降低了2×10³倍以上。虽然kon(CN-)的部分降低可以通过BHA.HRP复合物中铁的水配位来解释，但kon(CN-)和koff(CN-)的额外降低表明结合口袋中的BHA阻塞了CN-的配位通道，并充当了CN-交换的“门”。BHA结合后，HRP-CN中远端组氨酸不稳定质子与大量水的交换速率降低了4倍以上，这支持了这一观点。三元复合物BHA.HRP-CN表现出不均一性。虽然在两种三元复合物中BHA和CN-结合的热力学似乎相似，但两种复合物的BHA结合和解离速率相差约10倍。这两种不均一形式在25℃下以约2×10² s-1的速率相互转化，这使得通过饱和转移无法确定CN-结合速率的任何差异。两种三元复合物中其中一种的更高的不稳定性归因于一些远端残基的交替取向，这种取向在其中一种形式中阻塞了底物结合通道。三元复合物中从血红素到BHA的转移核Overhauser效应表明，BHA底物不仅与血红素吡咯D取代基接触，还与远端组氨酸42接触，这表明BHA的极性侧链很好地延伸到远端血红素口袋中。（摘要截短至400字）
