Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumour formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signalling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signalling, and that drugs designed to target this site may be potent anti-cancer agents.

Synthesis and evaluation of novel dipeptide-bound 1,2,4-thiadiazoles as irreversible inhibitors of guinea pig liver transglutaminase.

Herein we report the synthesis and evaluation of 14 novel peptides as potential irreversible inactivators of guinea pig liver transglutaminase (TGase). These peptides were designed to resemble Cbz-L-Gln-Gly, known to be a good TGase substrate, and to include a 1,2,4-thiadiazole group. The side chain length of the amino acid residue bearing the inhibitor group was also varied in order to permit investigation of this effect. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 0.330 to 0.89 microM(-1) min(-1).

Nonlinear free energy relationship in the general-acid-catalyzed acylation of rat kidney gamma-glutamyl transpeptidase by a series of gamma-glutamyl anilide substrate analogues.

The gamma-glutamyl transpeptidase (GGT) purified from rat kidney reacts with a series of eight parasubstituted L-glutamyl gamma-anilides, in the presence of Gly-Gly, catalyzing the formation of gamma-Glu-Gly-Gly (pH 8.0, 37 degrees C). The transpeptidation reaction was followed through the discontinuous colorimetric determination of the concentration of released parasubstituted aniline. Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each anilide substrate. A Hammett plot constructed by the correlation of log(k(cat)) and the sigma(-) parameter for each anilide substrate displays statistically significant upward curvature, consistent with a general-acid-catalyzed acylation mechanism in which the geometry of the transition state changes with the nature of the para substituent. Kinetic isotope effects were measured and are consistent with a reaction involving a proton in flight at the rate-limiting transition state. The pH-rate profiles measured over pH 7.0-9.5 are bell-shaped with kinetic pK(a) values that may be attributed to the active site nucleophile (or its general-base catalytic partner) and the active-site general acid. The variation of the latter pK(a) value as a function of temperature is consistent with an enthalpy of ionization expected for an ammonium ion acting as a general acid. Examination of the variation of k(cat) as a function of temperature gave values for the enthalpy and entropy of activation that are similar to those determined for the general-acid-catalyzed breakdown of the tetrahedral intermediate formed during acylation of chymotrypsin by similar amide substrates.

Kinetic studies of guinea pig liver transglutaminase reveal a general-base-catalyzed deacylation mechanism.

Guinea pig liver transglutaminase (TGase) reacts with 0.1 mM N-Cbz-L-Glu(gamma-p-nitrophenyl ester)Gly (5, prepared herein, K(M) = 0.02 mM) to undergo rapid acylation that can be followed spectrophotometrically at 400 nm (pH 7.0, 25 degrees C). Deacylation of the transiently formed thiolester acyl enzyme intermediate via catalytic aminolysis was studied in the presence of six primary amines of widely varying basicity (pK(NH+) = 5.6-10.5). Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each amine substrate. A Brønsted plot constructed through the correlation of log(k(cat)/K(M)) and pK(NH+) for each amine substrate displays a linear free-energy relationship with a slope beta(nuc) = -0.37 +/- 0.08. The shallow negative slope is consistent with a general-base-catalyzed deacylation mechanism in which a proton is removed from the amine substrate during its rate-limiting nucleophilic attack on the thiolester carbonyl. Kinetic isotope effects were measured for four acceptor substrates (water, kie = 1.1 +/- 0.1; aminoacetonitrile, kie = 5.9 +/- 1.2; glycine methyl ester, kie = 3.4 +/- 0.7; N-Ac-L-lysine methyl ester, kie = 1.1 +/- 0.1) and are consistent with a proton in flight at the rate-limiting transition state. The active site general-base implicated by these kinetic results is believed to be His-334, of the highly conserved TGase Cys-His-Asp catalytic triad.

Evaluation of novel dipeptide-bound alpha,beta-unsaturated amides and epoxides as irreversible inhibitors of guinea pig liver transglutaminase.

Herein, we report the results of irreversible inhibition of guinea pig liver transglutaminase (TGase) by a series of 24 novel dipeptides containing either an alpha,beta-unsaturated amide or an epoxide functional group. Their inactivation rate constants were measured using a direct continuous spectrophotometric method and were found to vary between 421 x 10(3) and 3000 x 10(3)M(-1)min(-1).

A direct continuous spectrophotometric assay for transglutaminase activity.

Herein we report the development of a direct and continuous spectrophotometric method for determining transglutaminase (TGase) activity by using N,N-dimethyl-1,4-phenylenediamine (DMPDA) as a gamma-glutamyl acceptor substrate and carbobenzyloxy-l-glutamylglycine (Z-Gln-Gly) as a typical peptide gamma-glutamyl donor substrate. The transamidation activity of TGase can thus be followed by monitoring the increase of absorbance of the resulting anilide product at 278 nm. The extinction coefficient of the authentic, independently synthesized anilide was determined to be epsilon = 8940 +/- 55 M(-1) cm(-1). Using this assay, we determined the apparent K(M) of DMPDA to be 0.25 mM, which compares favorably to the apparent K(M) values determined for other acceptor substrates under conditions where Z-Gln-Gly is also used as the donor substrate, such as N-acetyl-l-lysine methyl ester (9.6 mM) and methylamine (13.1 mM). Finally, the sensitivity of this assay technique was established through the measurement of irreversible inhibition constants for iodoacetamide, determined to be K(I) = 75 +/- 11 nM and k(inact) = (120 +/- 1) x 10(5) M(-1) min(-1).

Guinea pig liver transglutaminase: A modified purification procedure affording enzyme with superior activity in greater yield.

Tissue transglutaminase purified from guinea pig livers has a very broad substrate specificity in comparison with other members of the transglutaminase family and therefore is useful for substrate analogue kinetic studies. Modifications made in our laboratory to the standard purification protocol (J. E. Folk and S. I. Chung, 1985, Methods Enzymol. 113, 358-364) have yielded a 28% increase in specific activity and 55% increase in overall yield, while reducing the number of steps to the purification. Herein we report some of the highest yields and specific activities for guinea pig liver transglutaminase found in the literature, as well as the use of lyophilization as a solution to the long-standing problem of enzyme stability during storage.

Synthesis and characterization of a series of novel glutamic gamma-15N-anilide dipeptides.

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.

Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer.

The sodium-potassium adenosinetriphosphatase of sheep kidney, preincubated with sodium and magnesium (E.Nae), reacts with 0.01-2.00 mM ATP to form covalent phosphoenzyme (E-P). The first order rate constant for phosphorylation increases hyperbolically with ATP concentration with a maximum value of (4.6 +/- 0.9) x 10(2) s-1 and K0.5 = 75 +/- 25 microM (ph 7.4, 25 degrees C, 120 mM NaCl, and 3 mM MgCl2). If the phosphoryl-transfer step were rate-limiting, the approach to equilibrium to give 50% E-P in the presence of ADP would follow kobsd=Kf+Kr+9.2 x 10(2) s-1. However, the formation of phosphoenzyme from E.Na3 with 1.0 mM ATP plus 2.0 mM ADP proceeds to 50% completion with kobsd=(4.2 +/- 0.8) x 10(2) s-1. This result show that phosphoryl transfer from bound ATP to the enzyme is not the rate limiting step for phosphoenzyme formation from E.Na3. The result is consistent with a rate-limiting conformational change of the E.Na3.ATP intermediate that is followed by rapid phosphoryl transfer, with kcat > or = 3000 s-1.