A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4. 10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated deriv atives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMB O J. 14, 3855-3863]. This catalytic diad has been observed directly by prot on NMR. The single low-field line in the H-1 NMR spectrum is assigned by si te-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine H-delta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and e xchanges slowly with H2O on the NMR time scale. The position of the low-fie ld peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, refle cting a pK(a) of 8.0 at 6 degreesC, which is identified with the pK(a) of H is32. The H-delta1 signal is modulated by rapid exchange of the H-is an ele ment of2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR b ehavior is remarkably similar to that of the serine proteases. The postulat ed function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of p hosphatidylinositol (PI) substrate to form a catalytic triad analogous to A sp-His-Ser of serine proteases. This is an example of substrate-assisted ca talysis where the substrate provides the catalytic nucleophile of the triad . This hydrogen bond becomes shorter as the imidazole is protonated, sugges ting it is stronger in the transition state, contributing further to the ca talytic efficiency. The hydrogen bond fits the NMR criteria for a short, st rong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 Angstrom at pH 6 measured by NMR, a D/H fractionation fac tor significantly lower than 1.0, and a protection factor greater than or e qual to 100.