Carbon-13 nuclear magnetic resonance probe of active-site ionizations in human carbonic anhydrase B.

摘要: Human carbonic anhydrase B (HCAB), prepared by a new affinity chromatography procedure, was carboxymethylated exclusively at NT of its active-site histidine-200 using 90% [1-13C]bromoacetate. The 13C nuclear magnetic resonance signal the covalently attached carboxylate easily detected over natural abundance background due to other carbonyl and carboxyl carbons this 29 000 molecular weight zinc metalloenzyme. Its chemical shift proved very sensitive presence inhibitors in active site variations pH. Two perturbing groups with pKa values 6.0 9.2 were assigned modified itself zinc-bound water ligand, respectively, making use NMR titration data on Nr- Nr-carboxymethyl-L-histidine model compounds. results rule out as critical group whose ionization controls catalytic activity. They also strongly suggest an interaction carboxymethyl either or ligand around pH 8, possibly explaining basis for major differences between HCAB CmHCAB.