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A fluorescent protein-labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.

Conformational analysis: Capillary electrophoresis (CE) allows for the rapid separation of slowly interconverting protein conformers. Kinetic analysis (k(open), k(closed), and K(d)) of electropherograms in the presence and absence of effector ligands allows the measurement of kinetic and thermodynamic constants associated with conformational changes and ligand binding.

Novel amphiphilic aminoglycosides are shown to inhibit clinically relevant deactivating enzymes, without undergoing significant deactivation themselves.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite advances in therapies, patients with GBM still have a 14 month median survival rate after treatment. A common...

The enzyme γ-glutamyl transpeptidase (GGT) is implicated in cellular detoxification, the biosynthesis of leukotrienes and control of the physiological concentration of glutathione. It also plays important roles in Parkinson's disease, diabetes, apoptosis inhibition and cancer drug resistance. It catalyses the breakdown of its in vivo substrate, glutathione, by cleaving the amide bond between the γ-glutamyl and the cysteinylglycine moieties. Threonine is proposed to act as the nucleophile of GGT in the formation of the γ-glutamyl acyl enzyme intermediate during the acylation step. The γ-glutamyl moiety is then transferred to a primary amine acceptor substrate (an amino acid or dipeptide) or to a water molecule, to form a compound containing a new isopeptide bond or glutamate in the transamidation or hydrolysis reactions, respectively. In spite of the importance of the role of GGT in human physiology, there is a lack of information about the mechanisms of its catalytic reactions, and in particular the nature of the intermediate formed during the acylation step. In order to gain insight into the formation of the acyl enzyme intermediate, different D-γ-glutamylanilides substituted in the para position with electron-withdrawing and electron-donating groups were used as donor substrates under conditions where water served as acceptor substrate. A Hammett plot with a slope of zero was obtained for the steady-state hydrolysis reaction for which deacylation is the rate- limiting step. To confirm the ping-pong mechanism, pre-steady-state kinetics of this reaction were then performed with the donor substrate D-γ-glutamyl-p-nitroanilide, which liberates the chromophore p-nitroaniline. Experiments using a stopped- flow spectrometer and a rapid mix–quench apparatus gave biphasic traces with a burst up to ∼65 ms, the amplitude of which corresponds well with the concentration of the enzyme. These burst kinetics were also observed in the presence of L-methionine at concentrations ∼15-fold below its KM value, where deacylation would still be rate limiting. These observations are consistent with the formation of an intermediate during the rapid acylation step and support the modified ping-pong mechanism proposed for GGT- mediated hydrolysis and aminolysis. Copyright © 2004 John Wiley & Sons, Ltd.

Transglutaminases (TGases, EC 2.3.2.13) are enzymes that catalyze the cross-linking of peptides and proteins by the formation of isopeptide bonds between the γ-carboxamide group of a glutamine side chain and the ɛ-amino group of a lysine side chain. This reaction is known to occur via a modified ping-pong mechanism (Folk 1969) in which a glutamine-containing protein or peptide, the acyl-donor substrate, reacts with the enzyme’s catalytic cysteine residue to form a thioester bond which generates the covalent acyl-enzyme intermediate with concomitant release of ammonia (Scheme 1 ▶). This intermediate then reacts with a second substrate, the acyl-acceptor, which can be almost any primary amine (Aeschlimann and Paulsson 1994), to yield the amide product and free enzyme. The acyl-enzyme intermediate can also be hydrolyzed in the absence of primary amine but at a slower rate. A conserved Cys-His-Asp catalytic triad that is similar to that of cysteine proteases catalyzes these reactions. Although TGases exhibit high specificity towards the side chain of L-Gln as the acyl-donor substrate (Asn and D-Gln are not recognized), their specificity towards the acyl-acceptor is lower, and many primary amines can be recognized (Clarke et al. 1959; Folk 1983). Scheme 1. Catalytic mechanism of tissue TGase. TGases are divided into nine classes (Chen and Mehta 1999), of which the most abundant class is the ubiquitous tissue transglutaminase (Fesus and Piacentini 2002) found in all vertebrates (Wada et al. 2002). Tissue TGases are intracellular, monomeric enzymes that exhibit a calcium-dependent transglutaminase activity that is inhibited by GDP/GTP binding. Their structure comprises four domains (Fig. 1 ▶): the N-terminal β-sandwich domain, the catalytic core, the barrel 1 domain, and the C-terminal barrel 2 domain. Two crystal structures of tissue transglutaminase have been deposited in the Brookhaven Protein Data Bank. These are the structures of red sea bream tissue TGase (1G0D; Noguchi et al. 2001) and of human tissue transglutaminase with GDP bound at its allosteric binding site (1KV3; Liu et al. 2002). However, no TGase has yet been cocrystallized with ligand bound at the active site, limiting our understanding of the mode of substrate binding, although kinetic experiments have been performed that give clues as to the structural requirements for acyl-donor and acyl-acceptor substrates of tissue TGase (Clarke et al. 1959; Folk and Chung 1973). Folk and Cole (1965) made numerous observations with respect to the structural requirements for small peptide acyl-donor substrates of guinea pig liver TGase. They reported that Gln alone does not act as a substrate, nor do the peptides Gln-Gly and Gly-Gln-Gly. However, CBz-Gln-Gly, CBz-Gln-Gly ethyl ester, and benzoyl-Gly-Gln-Gly act as substrates. CBz-Gln shows poor but detectable activity. Folk and Cole concluded that for a glutamine on a small peptide to be recognized by tissue TGase it must be at least the next-to-last residue and the peptide should bear an N-terminal CBz group. For this reason, the commercially available CBz-Gln-Gly peptide serves as one of the most common nonproteic acyl-donor substrates for TGases in the literature. Figure 1. Crystal structure of red sea bream TGase (Noguchi et al. 2001). (A) Red sea bream TGase with active site residues circled. Front view. The P356–G369 loop is represented in ribbon diagram. (B) Top view. Although there is no consensus as to their physiological role, tissue TGases have been reported to act in endocytosis (Abe et al. 2000), apoptosis (Huo et al. 2003), extracellular matrix modification (Priglinger et al. 2003), and cell signaling (Singh et al. 2003). Tissue TGases have also been implicated in various physiological disorders such as Alzheimer’s disease (Kim et al. 1999), cataract formation (Shridas et al. 2001), and more recently, in Celiac sprue (Shan et al. 2002). As a result, development of inhibitors specific to tissue TGases (as opposed to type 1 or 3 TGases; Chen and Mehta 1999) is of current interest. However, to aid in inhibitor design, we need to gain a better understanding of the mode of binding of model peptide substrates to the enzyme. To this end, we performed a combined experimental/molecular modeling strategy consisting of (1) the synthesis of a series of CBz- or Boc-derivatized peptides and the measurement of their KM values, and (2) molecular modeling of their binding at the active site. Correlating the kinetic results with molecular modeling of the ligands bound at the active site cavity has allowed us to gain a better understanding of the requirements for productive binding of small peptide acyl-donor substrates.

The preparation of a series of novel Cbz-Gln-Gly dipeptide derivatives is reported, wherein the gamma-carboxamide groups of the glutamine side chains have been modified to gamma-15N-anilides which are substituted in the para position with -NO2, -Cl, -H, -CH3, -OCH3, and -N(CH3)2. Characterization of the free anilines (p(kappa)a values and 15N NMR chemical shifts) and corresponding gamma-anilides (15N NMR chemical shifts and FTIR wavenumbers) is also reported. Correlation of these physicochemical data to Hammett substituent parameters ((sigma)para) is discussed. These novel dipeptide derivatives should prove to be generally useful for structure-function enzymology studies of gamma-glutamyl transferring enzymes.

Treatment of a series of para-substituted aromatic and primary aliphatic carboxamides with NBS and NaOMe in methanol heated to reflux for ten minutes results in the conversion of the carboxamides to their corresponding primary amino methyl carbamates in nearly quantitative yields. The mild oxidative conditions of this modified Hofmann rearrangement are shown to be particularly useful for the preparation of p-substituted anilines.

The regioselectivity of the reaction of aniline with a series of N-protected aspartic and glutamic acid anhydrides was controlled through the choice of reaction solvent. In benzene, the formation of aspartic or glutamic acid α-anilides was favored, with α:(β or γ) regioselectivities as high as 100:0. On the other hand, in DMSO, the formation of either aspartic acid β-anilide or glutamic acid γ-anilide was favored, with α:(β or γ) regioselectivities as high as 0:100. This regioselectivity was not observed for alkylamines, amino acids, thiols, or alcohols. However, the high yields of the aniline reactions and the ability to specify their regioselectivities demonstrate the usefulness of our method for the formation of aspartic and glutamic acid anilide bonds.

Herein we report the synthesis of 24 novel peptides as potential irreversible inactivators of transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include either alpha,beta-unsaturated amide groups or the corresponding epoxide groups. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect.