Solid-state carbon-13 NMR study of a transglutaminase-inhibitor adduct

Abstract

We have used solid-state 13C NMR to study the structure of the adduct resulting from the inactivation of the enzyme transglutaminase by 3-halo-4,5-dihydroisoxazoles. These inhibitors were conceived on the assumption that they would inhibit transglutaminase by attack of an enzyme active site cysteine thiol on the imine carbon of the dihydroisoxazole ring. The tetrahedral intermediate formed could then break down with the loss of the halide group and the subsequent formation of a stable imino thioether adduct. We have compared the 13C CPMAS spectra of the chloro-, bromo-, and (ethylthio)dihydroisozazole inhibitors, and the results indicate that the chemical shift of the C-3 carbon is sensitive to the nature of the heteroatom. Subtraction of the natural-abundance 13C solid-state NMR spectrum of the enzyme from that of the enzyme inactivated by C-3-labeled chlorodihydroisoxazole reveals a broad peak at 156 ppm. The chemical shift of this peak is very close to that observed for a model 3-ethylthio compound and suggests the formation of a stable imino thioether enzyme adduct. Similar results were obtained for lyophilized enzyme adducts and for frozen solutions of the enzyme adduct in the absence and presence of Ca2+. We have also compared these results with those obtained by solution NMR on an aqueous solution of the enzyme-inhibitor complex. The 13C-labeled C-3 resonance was not observed in this case.

Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae

“…Larvae were scored live when they stained blue uniformly along their entire length and dead when the larvae remained unstained. Three TGase inhibitors were used: MDC, a competitive pseudosubstrate that acts as an inhibitor of endogenous protein cross-linking by the enzyme and CS, an active-site inhibitor, both known to be specific for TGase (13,25,40), and a new synthetic inhibitor of TGase, N-benzyloxycarbonyl-D,L-␤-(3-bromo-4,5-dihydroisoxazol-5-yl)-alanine benzylamide (kindly provided by Allen Krantz, Syntex Inc., Palo Alto, Calif.), which specifically inactivates mammalian TGases by binding to the active site of TGase (2,4). The results shown for each inhibitor concentration are the average number of larvae that molted or the average number of larvae that were scored alive in 5 to 10 wells containing a total of 50 to 100 L3.…”

Carbon and Nitrogen Chemical Shifts of Solid State Enzymes

“…For the case of 13 15 N chemical shift tensors of interesting chemical classes typically found in proteins. Open squares represent CSA elements δ 11 , δ 22 , and δ 33 , from left to right, respectively, and closed squares represent the isotropic chemical shift, δ iso . The square around δ 22 of the carbonyl group indicates the variation of that tensor element, which is caused by hydrogen bonding.…”

“…The anisotropy, or distribution of chemical shift values as a function of the spatial orientation of the sample in the magnetic field, can be measured in the solid state and is compactly summarized in the form of three values for three principal directions, δ 11 , δ 22 , and δ 33 . These three values of the chemical shift anisotropy (CSA) are usually measured by simulation of the magic angle spinning spectra taken at low spinning speeds.…”

Transglutaminase activity in equine strongyles and its potential role in growth and development

“…For this purpose, CS that inactivates the enzyme acti vity by binding to the active site and MDC, a compe titive amine substrate inhibitor that inhibits specifically the endogenous protein crosslinking by TGase (Lorand et al, 1979;Folk, 1980;Castelhano et al, 1990;Auger et al, 1993) were used at millimolar concentration. The results indicate that neither inhibitor affects larval via bility of S. vulgaris and S. equinus.…”

“…Effect on L4 viability with MDC and growth with both the inhibitors strongly supports the notion that TGase is involved in growth and development of these nematodes. N-bezyloxycarbonyl-D,L-b-(3-bromo-4,5-dihydroisoxazol-5-yl)-alanine benzylamide (Syntex Inc., Palo Alto, CA), ano ther TGase inhibitor that like CS binds to the active site of TGase (Auger et al, 1993;Castelhano et. al, 1990), also inhibited О. volvulus larval molting (Lustigman et al, 1995) and Nippostrongylus brazi liensis L4 viability and molting to adult (Castelhano et al 1990).…”