Catalytic mechanism of serine proteases: reexamination of the pH dependence of the histidyl 1J13C2-H coupling constant in the catalytic triad of alpha-lytic protease.
L-Histidine, 90% 13C enriched at the C2 position, was incorporated into the catalytic triad of alpha-lytic protease (EC 3.4.21.12) with the aid of histidine-requiring mutant of Lysobacter enzymogenes (ATC 29487), and the pH dependence of the coupling constant between this carbon atom and its directly bonded proton was reinvestigated. The high degree of specific 13C isotopic enrichment attainable with the auxotroph permits direct observation and measurement of this coupling constant in proton-coupled 13C NMR spectra at 67.89 MHz and at 15.1 MHz. In contrast to the earlier study, the present study indicate that this coupling constant does respond to a microscopic ionization with pKa near 7.0; moreover, the magnitude of the values of 1JC-H observed are in accord with those expected for titration of the histidyl residue. We conclude that the original measurement must be in error and that this coupling constant now also supports a histidyl residue that titrates more or less normally as a component of the catalytic triad of serine proteases.
D002384:Catalysis; D010450:Endopeptidases / Q000378:metabolism*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D012697:Serine Endopeptidases
D006639:Histidine; D010450:Endopeptidases; D012697:Serine Endopeptidases; C021826:myxobacter alpha-lytic proteinase