Proton NMR spectroscopic studies of selenosubtilisin

Abstract

Anomalously low-field signals in 1H NMR spectra of serine proteases provide valuable information on the protonation state of the catalytic histidine residue. We have examined the pH dependence of the deshielded protons of three different oxidation states of selenosubtilisin, a semisynthetic selenoenzyme with significant peroxidase activity, in order to evaluate the influence of the selenium prosthetic group on the hydrogen-bonding network in the modified active site. In the spectra of the anionic seleninate and selenolate derivatives, two resonances were observed at 18.0 and 15.5/14.0 ppm, assigned respectively to the N delta 1 and N epsilon 2 protons of protonated His64. These signals were apparent from pH 4 to above pH 10, indicating that the negatively charged prosthetic group increases the stability of the imidazolium dramatically, raising its pKa by at least 3-4 pH units. In contrast, a neutral selenenyl sulfide species exhibits no deshielded proton signals at 18 ppm at any pH but has a weak signal at 14.1 ppm above pH 7 which was assigned to the N delta 1 imidazole proton of neutral His64. While the pKa of His64 appears normal (approximately 7) in this derivative, the selenenyl sulfide substitution may alter the orientation of the imidazole ring within the active site for steric reasons. Together with data on the influence of pH on peroxidase activity, these results suggest that selenosubtilisin's His64 acts as a general acid facilitating the reduction of the selenenyl sulfide to selenolate by thiols.

Differing views of the role of selenium in thioredoxin reductase

“…The crystal structure of selenosubtilisin shows that it is in the seleninic acid form (Enz-SeO 2 − ) (Syed et al 1993). NMR studies of selenosubtilisin show that it also exists in the selenenic acid (Enz-SeOH) and selenosulfide (Enz-Se-SR) forms (House et al 1993). If our hypothesis is incorrect, then the Enz-SeO 2 − form should be inactive, however, all three forms of selenosubtilisin have peroxidase activity (Bell et al 1993).…”

Selenopeptide chemistry

“…Generally it is well established that exposed selenols are a very reactive species and readily oxidised by air [110]. 1 H-NMR studies of selenoenzyme selenosubtilisin revealed that the enzyme-bound selenol and seleninic acid have unusually low pK A values when compared to typical selenium compounds and were found to be deprotonated at all accessible pHs [111]. Intriguingly, Sec residues in peptides are found to be fully oxidised directly upon deprotection via hydrogen fluoride [27] (Metanis N, Dawson PE.…”

Towards More Efficient Glutathione Peroxidase Mimics: Substrate Recognition and Catalytic Group Assembly

“…Although the proposed catalytic mechanism, which involves two cycles and six intermediates, is significantly different from that of GPX, the obvious enhancement of GPX-like activity observed by introducing an amino nitrogen near the divalent selenium, strongly indicates the importance of the amino groups located in the vicinity of the diselenide bond. Hilvert et al [91][92] have reported that an artificial enzyme selenosubtilisin, possessing a strongly basic histidine residue at the active center like Wilson's catalyst, exhibits high GPX-like activity. They concluded that the protonated histidine acts as a general acid facilitating the reduction of the selenenyl sulfide to selenolate with thiols.…”

“…The selenol, like the seleninic acid, is known to have an unusually low pKa value from spectroscopic titration of its 77 Se resonance, and its departure from the selenenyl sulfide is expected to be facilitated by protonation of the active site histidine. The resulting selenolate-imidazolium ion pair is quite stable over the pH range 4-10, and protonation of an active site residue (pK a≈7) has been shown to be necessary for efficient turnover catalysis [91][92].…”