PubMed Health⌕ Search

Biomedical subjects

I Kuntz

Publications and source records attributed to I Kuntz.

5 recordsLinked to original sources
PubMed2001

Definition of the extended substrate specificity determinants for beta-tryptases I and II.

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.

Humans↗
PubMed1989

Identification by proton nuclear magnetic resonance of the histidines in cytochrome b5 modified by diethyl pyrocarbonate.

Diethyl pyrocarbonate (DEP) is an electrophilic reagent that is used to modify reversibly the histidine residues of proteins. Unfortunately, the lability of the acylated histidine adduct usually does not permit the isolation and identification of the modified histidine. By use of 500-MHz proton NMR spectroscopy, it has been possible to identify the C-H resonances of the nonaxial histidines of trypsin-solubilized bovine, rabbit, and porcine cytochrome b5 and therefore observe the interaction of DEP with specific histidine residues of cytochrome b5. In addition, the pKa of the peripheral histidines of bovine and rabbit cytochrome b5 have been measured in D2O. In the bovine protein it was found that the histidines are modified sequentially with increasing DEP concentration in the order His-26 greater than His-15 greater than His-80. This order is maintained in the rabbit protein with the following additions: His-26 approximately His-27 greater than His-15 greater than or equal to His-17 greater than His-80. The relative reactivity of the peripheral histidines with DEP was rationalized by considering three of their characteristics: (1) the pKa of the histidine, (2) the fraction of the side chain exposed to the solvent, and (3) the hydrogen-bond interactions of the imidazole ring.

Animals↗
PubMed1975

Adenine nucleosides in solution: circular dichroism studies and base conformation.

Adenosine, AMP, S-adenosylhomocysteine, S-adenosylmethionine, aristeromycin and 25 other synthetic adenosine analogs modified in the 4' or 5' positions show certain groups of different circular dichroism (CD) spectra. Both positive and negative Cotton effects can occur in the long-wavelength part (250-270 nm) of the spectra. Molar ellipticities [theta] range from -6000 (in adenosine 5'-carboxylate) to +4000 deg. cm2 dmol-1 (in 5'-deoxy-5'iodoadenosine), including some compounds with small, polar 5'-substituents in which low-intensity bands are found in signed pairs. Most of these adenosine derivatives that have the same adenine chromophore and a ribofuranose moiety unsubstituted in the 2' and 3' positions prefer an anti-conformation of the adenine base, as evidenced by proton magnetic resonance spectroscopy. In the majority of cases, electronic perturbations of the chromophore or major alterations of the assymmetric sugar residue can be excluded as sources of the CD variations. Therefore a correlation of the long-wavelength CD bands with the glycosyl torsion angle phiCN is suggested, where the gauche, gauche/anti combination which is typical of AMP in the crystal and in solution (phiCN approximately -40degrees, [theta] negative) is one reference point and a region for phiCN = 0degrees ([theta] positive) is assigned to compounds with space-filling substituents such as S-adenosylmethionine. Both negative and positive Cotton effects can be associated with the anti conformation range. Within this series, the base conformation of novel nucleoside structures could be predicted from CD measurements. The CD spectrum gives no indication, however, of whether a certain torsion angle is the result of a rigid structure (as in AMP) or the average value of a molecule with high rotational freedom (as in 5'-deoxyadenosine). The conformations of aristeromycin and 4'-thioadenosine are discussed in relation to adenosine, and a structure-determining effect of the 4' bridge atom is noted.

Adenosine↗