Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.

The 2-amino alcohol structure -CH(NH2)CH(OH)- exists in proteins and peptides in N-terminal Ser or Thr and in hydroxylysine. Its very rapid oxidation by periodate at pH 7 generates an aldehyde in the peptide and is the first step in a method for site-directed labeling with biotin or a fluorescent reporter. The modifying group is a hydrazide, RCONHNH2, which reacts with the new aldehyde to form a hydrazone-peptide conjugate, RCONHN = CH-peptide. Experiments with two synthetic peptides, Ser-Ile-Gly-Ser-Leu-Ala-Lys and Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, and with recombinant murine interleukin-1 alpha (an 18-kDa cytokine with N-terminal Ser) demonstrated this method of peptide tagging. The use of a low molar ratio of periodate to peptide minimized the potential for side reactions during the oxidation, and the desired oxidation was rapid and highly specific. The hydrazones formed were stable at pH 6-8 for at least 12 h at 22 degrees C, but were labile at more acidic pH values. Potential uses of this method include the attachment of biotin, reporter groups, metal chelating groups, imaging agents, and cytotoxic drugs to peptides.

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Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme, and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.