Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase.
Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.
de Ropp
J S
JS
NMR Facility, Department of Chemistry, University of California, Davis 95616, USA.
Mandal
P K
PK
La Mar
G N
GN
D001565:Benzoates / Q000737:chemistry; D001665:Binding Sites; D003486:Cyanides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006877:Hydroxamic Acids / Q000737:chemistry; D046911:Macromolecular Substances; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D012996:Solutions; D013379:Substrate Specificity
D001565:Benzoates; D003486:Cyanides; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D046911:Macromolecular Substances; D012996:Solutions; D006418:Heme; D006639:Histidine; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid