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Titolo:
A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C
Autore:
Ryan, M; Liu, T; Dahlquist, FW; Griffith, OH;
Indirizzi:
Univ Oregon, Inst Mol Biol, Eugene, OR 97403 USA Univ Oregon Eugene OR USA 97403 egon, Inst Mol Biol, Eugene, OR 97403 USA Univ Oregon, Dept Chem, Eugene, OR 97403 USA Univ Oregon Eugene OR USA 97403 v Oregon, Dept Chem, Eugene, OR 97403 USA
Titolo Testata:
BIOCHEMISTRY
fascicolo: 32, volume: 40, anno: 2001,
pagine: 9743 - 9750
SICI:
0006-2960(20010814)40:32<9743:ACDIIS>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR-MAGNETIC-RESONANCE; BACILLUS-CEREUS; SERINE PROTEASES; IONIZATION-CONSTANTS; DIRECTED MUTAGENESIS; CRYSTAL-STRUCTURE; MODEL-COMPOUND; HISTIDINE; TRIAD; CHYMOTRYPSIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
49
Recensione:
Indirizzi per estratti:
Indirizzo: Griffith, OH Univ Oregon, Inst Mol Biol, Eugene, OR 97403 USA Univ OregonEugene OR USA 97403 l Biol, Eugene, OR 97403 USA
Citazione:
M. Ryan et al., "A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C", BIOCHEM, 40(32), 2001, pp. 9743-9750

Abstract

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4. 10) areubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diadat the active site of Bacillus cereus PI-PLC composed of aspartate-274 andhistidine-32 was postulated from the crystal structure to form a catalytictriad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the H-1 NMR spectrum is assigned by site-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 exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pK(a) of 8.0 at 6 degreesC, which is identified with the pK(a) of His32. The H-delta1 signal is modulated by rapid exchange of the H-is an element of2 with the solvent. Estimates of the exchange rate as a function ofpH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong 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 factor significantly lower than 1.0, and a protection factor greater than or equal to 100.

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Documento generato il 12/08/20 alle ore 19:43:20