Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes.
15N NMR spectroscopy was used to examine the active-site histidyl residue of alpha-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. With the better inhibitors of the class this arrangement is stable over the pH range 4.0-10.5. The results are consistent with a putative tetrahedral intermediate like complex involving a negatively charged, tetrahedral boron atom covalently bonded to O gamma of the active-site serine. (2) Boronic acids that are not substrate analogues form complexes in which N epsilon 2 of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N delta 1 of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. Benzeneboronic acid, which falls in this category, forms an adduct with histidine. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field 1H resonances and the 15N-H spin couplings. These results, coupled with the kinetic data of the preceding paper [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry (preceding paper in this issue)], indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.
Bachovchin
W W
WW
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111.
Wong
W Y
WY
Farr-Jones
S
S
Shenvi
A B
AB
Kettner
C A
CA
D001665:Binding Sites; D001897:Boronic Acids / Q000494:pharmacology*; D055598:Chemical Phenomena; D002621:Chemistry; D006639:Histidine; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000494:pharmacology*; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors
D001897:Boronic Acids; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D006639:Histidine; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase