Abstract
Serine proteases are one of the largest groups of enzymes, found in both eukaryotes and prokaryotes, and are responsible for many different functions. The detailed information about the hydrogen-bonds in the catalytic triad (Asp…His…Ser) of these enzymes is of importance in order to fully understand the mechanism of action. The aspartate of the triad is hydrogen bonded to the histidine but the exact nature of this bond has been under discussion for some time. It is either a common short ionic hydrogen bond (SIHB) or a delocalized low barrier hydrogen bond (LBHB) were the hydrogen bond is shorter. So far, the evidence for LBHB in proteins have not been conclusive. Here we show clear NMR evidence that LBHB does exist in NS3, a serine protease from Dengue. The one bond coupling constant between the hydrogen and nitrogen was shown to be only 52 Hz instead of the usual 90 Hz. This together with a 1H chemical shift of 19.93 ppm is evidence that the hydrogen bond distance between His and Asp is shorter than for SIHB. Our result clearly shows the existence of LBHB and will help in understanding the mechanism of the catalytic triad in the important group of serine proteases.
Highlights
The incidence of dengue virus (DENV) has grown around the world in recent decades and constitutes a major threat to human health
There remains the question whether the Asp...His hydrogen bond in the catalytic triad is associated with a normal short ionic hydrogen bond (SIHB)[3,4] or with a delocalized resonance effect called a low-barrier hydrogen bond (LBHB) or with a single well hydrogen bond (Fig. 1)[5,6,7]
The LBHB proponents and opponents have shown several experimental observations which could be categorized into the following groups: (a) unusual low-field chemical shift (CS) of protons involved in H-bonding[3,5,6,8] (b) measurement of H-bonding length by ultra-x-ray[10] (c) PK studies using 15N and 13C nucleus[11] (d) H/D isotope effect studies[12]
Summary
The incidence of dengue virus (DENV) has grown around the world in recent decades and constitutes a major threat to human health. Unusual large low- field shift of the Nδ1H protons, for single-well H-bonds (20–22 ppm) and for LBHB (17–19 ppm) has been proposed as one of the main criteria for the formation of a short, strong hydrogen bond under protonation of the catalytic His in complex with Asp in the intermediate induced complexes of serine type
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