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.

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Studies of the crystal structures of thymidylate synthase (TS) have revealed that a kink is present in beta-sheets that form the core of the enzyme. The beta-kink is proposed to serve as a "hinge" during conformational changes that occur in the enzyme after ligand binding at the active site. A residue in one of the beta-bulges that form the kink, glutamine at position 214 of human TS, is highly conserved in all TSs and is postulated to interact with nucleotide ligands that bind at the active site. To examine the role of this residue, glutamine at position 214 was replaced by residues that differ in volume, hydrophobicity, electrostatic charge, and hydrogen bonding potential. Genetic complementation studies utilizing a TS-deficient bacterial strain revealed that residues with large side chain volumes or that are prohibited in beta-bulges created loss of function proteins. Kinetic studies indicated that residue hydrophobicity is not correlated with catalytic activity. Residues that are predicted to alter the charge at position 214 created enzymes with kcat/Km values at least 10(3) lower than those of the wild type. Kinetic and ligand binding studies indicated that residue 214 is involved in nucleotide binding; however, hydrogen bonding potential does not contribute significantly to nucleotide binding energy. The data are consistent with the hypothesis that residue 214 is involved in maintaining the enzyme in a conformation that facilitates nucleotide binding and catalysis.

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13C chemical shifts and Jc,h coupling constants are reported for 17 5-substituted uracils. Overall, the chemical shifts at C-5 and C-6 of the 5-substituted uracils exhibit no obvious correlation with substituent electronegativity. Instead, when the 5-substituted uracils are considered as trisubstituted ethylenes, the chemical shift data are shown to be rationalized in terms of the ability of the C-5 substituent to behave as a mesomeric acceptor or donor. It is also demonstrated that the correlation of the chemical shifts at C-6 can be used to identify two categories of 5-substituted uracils whose parent deoxynucleotide derivatives are inhibitors of the enzyme, thymidylate synthetase. It is suggested that 13C nmr spectroscopy is a potentially useful tool for predicting the effectiveness of certain modified substrates as enzymatic inhibitors.Uracil and certain of its 5-substituted analogs are involved in a number of biochemically significant roles (i.e., constituents of RNA and DNA) in all(1) Presented in part at the 162nd National Meeting of the American