The extended active site of guinea pig liver transglutaminase.

The catalytic activities of guinea pig liver transglutaminase toward glutamine-containing peptide derivatives of three series have been studied. These series include: (a) formylheptapeptides of the basic structure, HCO-GLY3-L-Gln-Gly3. A single L-leucine residue was systematically substituted for glycine at a different position in each peptide; (b) formyltripeptides of the basic structure, HCO-Gly-L-Gln-Gly. L-Leucine was substituted for glycine in each position and in both positions; (c) various N-acyl derivatives of the dipeptide, L-Gln-Gly. Comparison of the values of the kinetic constants for methylamine incorporation and for hydroxylamine incorporation with the peptide derivatives shows that the length of the peptide chain has a pronounced influence on catalysis, as does the position of the leucine residue in the longer chain peptide derivatives. The kcat/Km(app) values for each substrate calculated from data for methylamine incorporation and from those for hydroxylamine incorporation were found to be in good agreement. However, both the observed maximum velocity and the apparent Michaelis constant for each peptide derivative were significantly larger for hydroxylamine incorporation than for methylamine incorporation. Interpretation of these findings as evidence for a normal catalytic mechanism for each amine incorporation reaction and for the limiting nature of deacylation to methylamine is discussed. Two observations caution against such an interpretation. These are the significantly higher inhibitor constants found fo formylhexaglycine and for several other competitive inhibitors in the hydroxylamine incorporation reaction, and earlier findings of higher turnover values with hyroxylamine in cases were acylation appears to be limiting for methylamine incorporation. Methods of preparation, supporting analytical data and properties of the peptide intermediates, the peptides, and their derivatives used in this study are presented in the miniprint supplement immediately following this paper.     

Specificity of guinea pig liver transglutaminase for amine substrates.

The amine specificity of guinea pig liver transglutaminase, a model enzyme for endo-gamma-glutamine:epsilon-lysin transferases, was explored with the aid of synthetic substrates of high apparent affinities. As exemplified by dansyl- (5-dimethylamino-1-naphthalenesulfonyl), (2,4-dinitrobenzenesulfonyl)-, and (2,4,6-triisopropylbenzenesulfonyl)-cadaverines--each of which showed affinities of approximately 4 x 10(7) M-1--the best amine substrates carried a large hydrophobic substituent attached to an alkylamine side chain of about 7.2 A in length. Altogether, our results point to the importance of a hydrophobic binding region in the enzyme from where the alkyl side chain reaches into a narrow crevice toward the active center and positions the primary amine of the substrate for attacking the carbonyl group of the acyl enzyme intermediate.     

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.     

Specific fluorescent labeling of chicken myofibril Z-line proteins catalyzed by guinea pig liver transglutaminase

Guinea pig liver transglutaminase has been found to catalyze the covalent incorporation of dansylcadaverine into chicken skeletal muscle myofibril proteins. Epifluorescence microscopy reveals that the incorporated dansylcadaverine is specifically localized at or near the myofibril Z line. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that actin constitutes a major fraction of the labeled material; the Z-line proteins alpha-actinin and desmin also show significant labeling, as well as tropomyosin, several additional unidentified proteins, and material with an extremely high molecular weight. The Z-line-specific fluorescence can be removed by brief trypsinization, which releases fluorescent alpha-actinin into the supernate. The majority of the fluorescent protein species are resistant to extraction by either 0.6 M KCl or KI. These results, in conjunction with the microscopic localization, suggest that the dansyl-labeled proteins are constituents of the myofibril Z line. A significant amount of fluorescently labeled transglutaminase is also present in labeled myofibrils, which is resistant to extraction with either 0.6 M KCl or KI. This result indicates a strong, noncovalent interaction between the transglutaminase molecule and the myofibril Z line.     

Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase

Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the K(m) value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase.     

Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate.     

Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutaminyl residues. These enzymes are involved in many biological phenomena. In this paper, the complete amino acid sequence of guinea pig liver transglutaminase, a typical tissue-type nonzymogenic transglutaminase, was predicted by the cloning and sequence analysis of DNA complementary to its mRNA. The cDNA clones carrying the sequences for the 5'- and 3'-end regions of mRNA were obtained by use of the sequence of the partial-length cDNA of guinea pig liver transglutaminase [Ikura, K., Nasu, T., Yokota, H., Sasaki, R., & Chiba, H. (1987) Agric. Biol. Chem. 51, 957-961]. A total of 3695 bases were identified from sequence data of four overlapping cDNA clones. Northern blot analysis of guinea pig liver poly(A+) RNA showed a single species of mRNA with 3.7-3.8 kilobases, indicating that almost all of the mRNA sequence was analyzed. The composite cDNA sequence contained 68 bases of a 5'-untranslated region, 2073 bases of an open reading frame that encoded 691 amino acids, a stop codon (TAA), 1544 bases of a 3'-noncoding region, and a part of a poly(A) tail (7 bases). The molecular weight of guinea pig liver transglutaminase was calculated to be 76,620 from the amino acid sequence deduced, excluding the initiator Met. This enzyme contained no carbohydrate [Folk, J. E., & Chung, S. I. (1973) Adv. Enzymol. Relat. Areas Mol. Biol. 38, 109-191], but six potential Asn-linked glycosylation sites were found in the sequence deduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic beta-glucosidase.

We demonstrate that although the guinea pig liver cytosolic beta-glucosidase does not catalyze the hydrolysis of gentiobiose, it does hydrolyze, disaccharide-containing glycosides such as p-nitrophenyl-beta-D-gentiobioside (Glc beta 1----6Glc beta-pNP) and mandelonitrile-beta-D-gentiobioside (amygdalin). Furthermore, we establish that the enzyme attacks disaccharide glycosides exolytically; specifically, we document the exolytic deglucosylation of amygdalin and the generation of the intermediate monosaccharide glycoside mandelonitrile-beta-D-glucoside prior to the formation of the aglycone (mandelonitrile). We also show that the cytosolic beta-glucosidase catalyzes the hydrolysis of various phenolic (e.g. arbutin and salicin) and cyanogenic plant glucosides (e.g. prunasin). Using the everted gut-sack technique, we demonstrate that the plant glucosides, amygdalin, prunasin, and vicine, are transported across the small intestine of the guinea pig efficiently and without being hydrolyzed. Based on these data we speculate that the cytosolic beta-glucosidase may participate in biotransformation of toxic plant glucosides.     

Purification of guinea pig liver transglutaminase using a phenylalanine-sepharose 4B affinity column

Guinea pig liver transglutaminase has been purified utilizing an improved protocol. The new steps include QAE-Sephadex ion-exchange, hydroxylapatite adsorption, and affinity chromatography using a phenylalanine-Sepharose column. The overall yield for the enzyme was 31%. This new scheme should enable more laboratories to take advantage of the protein crosslinking and labeling properties of transglutaminase.     

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 Br?nsted 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.     

Effects of calcium on the stability and activity of guinea pig liver transglutaminase

The binding of calcium to transglutaminase was studied by a kinetic method and by spectrophotometric titration. By the first method, we have shown that the binding of a single calcium per molecule, with a binding constant of 7500 +/- 1300 M-1 (at 55 degrees C), was responsible for the enhancement of the thermostability. The kinetic constants of the deactivation for the unliganded native form and the liganded native one are 1.47 +/- 0.04 min-1 and 0.32 +/- 0.05 min-1 respectively. The enhancement of thermostability is due to the stabilization of the native form, since the deactivation constants of the liganded and unliganded intermediate forms are equal. The total number of calcium binding sites, determined by titration is 4, and therefore, only 1 of them should be implicated in the thermostability. The 4 apparent association constants have been determined by a non-linear fitting of the data to the Adair equation. We have also shown a positive co-operative behaviour of the transglutaminase when the transferase is monitored versus calcium concentration.     

Interaction of liver plasma membranes and GTP with GTP hydrolysis

[¹⁴C]GTP or a metabolic product of GTP binds to liver membranes. Less label was associated with membranes when membranes were incubated with increasing concentrations of carrier GTP; ATP did not displace the label. Chromatography of extracted incubation mixtures of [¹⁴C]GTP and membranes revealed that over 96% of the nucleotide was hydrolyzed to 5′GMP and guanosine, Exposure of liver membranes to GTP prevented the separation of characteristic membrane bands that could be obtained when centrifugation was carried out without GTP. These studies indicate that GTP-effected alteration of liver plasma membranes is concomitant with GTP hydrolysis. These effects may be in addition to direct effects of GTP on enzymes and membrane proteins.     

Inhibition of erythrocyte transglutaminase by GTP

The guanine nucleotides GTP, GDP and GMP inhibit the activity of erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) in a decreasing order of effectiveness. The inhibition is more apparent at low than at saturating levels of calcium ions and is not due to the chelation of Ca2+, but to an interference with the process of activation by the cation. This inhibition is likely to contribute to the latency of erythrocyte transglutaminase in physiological conditions.