Friedhelm Schneider

Chemical investigations on pig kidney aminoacylase

1. Preparations of purified pig kidney aminoacylase (N-Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. It is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.

Cytostatischer Wirkungsmechanismus der Methylhydrazine

Cytostatic active methylhydrazine derivatives split off formaldehyde after mild dehydrogenation with potassium hexacyanoferrate (III). It is suggested that the primary formedazomethines andN-hydroxymethylderivatives participate in cytostatic efficacy by their alkylating ability (aminomethylation). Formaldehyde alone and its condensation products with hydrogen peroxide especially bis-hydroxymethylperoxide may exert cytostatic effects too.

The pH-Dependence of the Peptidase Activity of Aminoacylase

Aminoacylase is a potent peptidase around pH 8.5. The pH dependence of the Km values reveals that only dipeptides with uncharged N-terminal amino acids are substrates of the enzyme. The Km values reflect the hydrophobicity of the N-terminal amino acids. Calculated on the basis of unprotonated peptides they are pH independent. Hydrophobic, deprotonated amino acids are competitive inhibitors of the enzyme, tryptophan and norleucine being the strongest inhibitors. Inhibitor constants with glycylalanine as substrate have been determined for several amino acids. From the present results it may be deduced that the N-terminal amino acids of dipeptides are bound at a strongly hydrophobic site.

Cytostatischer Wirkungsmechanismus von Natulan

Hydrogen peroxide and formaldehyde, both products of catabolism of “Natulan”, inhibit DNA polymerase as well as DNA dependent RNA polymerase, as shown in the cell-free system. Formaldehyde, furthermore, interferes with the synthesis of nucleoside triphosphates. N-hydroxymethyl compounds which may be obtained from the breakdown of “Natulan” act like formaldehyde. In addition, there is a strong evidence that they react directly with DNA. Formalhydrazine, an azomethine derivative, is another basic compound of “Natulan” catabolism. It reacts with thiol compounds at 20°C and pH 7.2. The same reactivity, as displayed by the N-hydroxymethyl compounds, is observed when it is tested in the DNA and RNA polymerase systems. 14C-Uridine incorporation by lymphatic leukemic cells was inhibited by “Natulan”, azo-Natulan (dehydrogenated Natulan), 4-formyl-N-isopropyl-benzamide and 4-hydroxymethyl-N-isopropyl benzamide. The two latter compounds reduced the intracellular concentration of the nucleosides, suggesting blockage of nucleoside transport into the cells. This effect has been assigned to the substitution of N-isopropylcarbamide on the benzyl residue. The favorable therapeutic action of Natulan in lymphogranulomatose and other lymphoreticular neoplastics could be due therefore to its specific inhibition of nucleoside transport into the lymphocytes which are dependent on obtaining these compounds from the medium. Am zellfreien System wird gezeigt, daß Hydrogenperoxyd und Formaldehyd—beide als Abbauprodukte von Natulan nachgewiesen—die DNS-Polymerase und die DNS-abhängige RNS-Polymerase hemmen. Formaldehyd hemmt außerdem die Bildung der Nucleosid-triphosphate. N-Hydroxymethyl-Verbindungen, wie sie im Natulan-Abbau entstehen können, wirken wie Formaldehyd; greifen darüber hinaus aber die DNS direkt an. Formalhydrazin, Grundkörper eines im Natulan-Abbau auftretenden Azomethinderivates, reagiert bei 20° und pH 7,2 mit Thiol-Verbindungen. Gegenüber DNS- und RNS-Polymerase verhält sich Formalhydrazin wie eine N-Hydroxymethyl-Verbindung (s. o.). An lymphatischen Rinderleukosezellen wird gezeigt, daß Natulan, Azo-Natulan (=dehydriertes Natulan), dessen Abbauprodukt 4-Formyl-N-isopropyl-benzamid sowie 4-Hydroxymethyl-N-isopropyl-benzamid den Einbau von markiertem Uridin hemmen. Am Beispiel der beiden letzteren Verbindungen wird nachgewiesen, daß es zu einer umfassenden Senkung der intracellulären Nucleosid-Konzentration kommt, da der Eintritt der Nucleoside in die Zellen gehemmt ist. Der Effekt beruht auf der Substitution des Benzylrestes mit N-Isopropyl-carbonamid. Die günstige therapeutische Wirkung von Natulan bei Lymphogranulomatose und anderen lymphoretikulären Neoplasien wird damit erklärt, daß Lymphocyten, die auf Zufuhr von Nucleosiden aus dem Medium angewiesen sind, durch die Hemmung der Nucleosidaufnahme unter Natulan spezifisch geschädigt werden.

On the substrate specifity of l-asparaginase from E. coli

Recently the question was discussed whether L-asparaginase (EC3.5.1.1) from Escherichiu coli, and other asparaginases with anti-tumor activity may in vivo exert activities other than the hydrolysis of L-asparagine. For example the deamidation of fetuin [l] and L-asparaginyl-tRNA [2] by asparaginase was observed. Furthermore asparaginase from Erwinia carotouoru was reported to enhance the velocity of deamidation of some peptides with COOH-terminal asparagine [3]. We therefore extended our studies on the substrate specifity and kinetics of E.coli asparaginase [4] on a series of NZand N4 -substituted asparagine derivatives and some other substrate analogues not yet tested with the E.coli enzyme.

Renal aminoacylase, a zinc enzyme.

Renal aminoacylase is inactivated by dialysis against metal complexing agents such as o-phenanthroline. Activity can be restored by addition of zinc ions. A zinc dissociation constant of about 10-10 м at pH 7.8 is obtained by titration of the enzyme with a metal ion buffer. The reactivity of the SH groups of the enzyme is considerably affected by zinc ions. The enzyme contains two essential zinc ions per molecule.

Chemical modification of one carboxyl-group of papain abolishes the catalytic activity of the enzyme

The participation of a carboxyl-group in the catalytic mechanism of papain was postulated for the first tune by Smith in 1954 [ 11. The papain mechanism proposed by Smith was based mainly on kinetic data. Later investigators have refused the function of a carboxyl-group in the catalytic process and have given experimental evidence for the implication of an imidazole residue in the catalytic cycle [2,3] ; this concept seemed to be supported by the results of X-ray analysis of the enzyme [3]. Nevertheless there are some objections to the involvement of an imidazole residue in the catalytic process concerning especially the pK-values [4,5]. The pH-profile of the catalysis has led to the postulation that the apparent pK of the respective imidazolyl group must be abnormally high, being as high as 9.8-10 [3] or as low as 4 [2]. Our modification experiments of the enzyme with diazo-I-H-tetrazole [6] as well as the results of photo-oxidation [4] do not give any indications on an imidazolyl-group with an abnormal pK-value. We therefore have undertaken a renewed investigation of the role of the carboxylgroups of papain using chemical modification with a nucleophil and carbodiimid [7]. In the present communication we describe the results of these experiments which demonstrate the loss of catalytic activity after chemical modification of only one carboxylgroup of the enzyme.

Vergleichende Untersuchung einiger physikalisch-chemischer Verfahren zur quantitativen Analyse von SH-Verbindungen verschiedener Struktur

Some known methods for the determination of SH-groups are investigated with respect to their applicability, reliability an susceptibility to interferences. For this purpose comparative determinations of 18 compounds of different structure are carried out. Amperometric titration with AgNO3 at pH 7.4 yields correct results for certain types of compounds only. For many substances the results are reproducible but considerably exceed the expected stoichiometry. Polarographic determinations in 0.6 M HClO4 produce linear calibration plots. The steepness of these plots, however, depends on the size of the molecules. Photometric determinations according to Ellman or Boyer yield reliable results. Both methods require calibration by a substance of known SH-content. Potentiometric measurement with the Ag/AgI-electrode after reaction with iodoacetate is the most suitable method for the determination of absolute SH-content; the results were highly reproducible and resiable for all compounds investigated.ZusammenfassungEine Reihe bekannter Bestimmungsmethoden für SH-Gruppen wird auf Anwendbarkeit, Zuverlässigkeit und Störanfälligkeit geprüft. Zu diesem Zweck werden je 18 Verbindungen unterschiedlicher Struktur vergleichend analysiert. Amperometrische Titration mit AgNO3 bei pH 7.4 führt nur bei einigen Verbindungsklassen zu richtigen Ergebnissen. Mit vielen Substanzen ergeben sich reproduzierbare, aber überstöchiometrische Werte. Polarographische Analyse in 0.6 M HClO4 führt zu linearen Eichdiagrammen, deren Steigung von der Molekülgröße abhängt. Photometrische Bestimmung sowohl nach Ellman als auch nach Boyer führt zu verläßlichen Ergebnissen. Beide Methoden bedürfen der Eichung durch eine Substanz mit bekanntem SH-Gehalt. Potentiometrische Bestimmung mit der Ag/AgJ-Elektrode nach Umsetzung mit Jodacetat ist die vorteilhafteste Methode zur Absolutbestimmung des SH-Gehaltes und führt mit allen untersuchten Verbindungen zu gut reproduzierbaren und zu verlässigen Ergebnissen.

Identification of essential histidine residues of aminoacylase by photooxidation and by reaction with diethylpyrocarbonate.

Abstract State and function of the histidine residues of aminoacylase were investigated by photooxidation in the presence of methylene blue and by chemical modification with diethylpyrocarbonate. Complete inactivation of the enzyme was observed after oxidation of 4 histidine residues. From the pH dependence of the photooxidation it becomes evident that the inactivation of the enzyme is not a consequence of the simultaneous oxidation of tryptophan residues. The enzyme is also inctivated by chemical modification of histidine residues with diethylpyrocarbonate. Activity is restored by treatment with hydroxylamine. Zn2+-ions which are essential for the activity of amino acylase protect the available histidine molecules against photooxidation and attack by diethyl pyrocarbonate. It is suggested that histidine is involved in the binding of the essential Zn2+-ions.

Catalytic properties of peptides with hydrolytic activity

The functional groups of the amino acids are the tools of catalysis in those enzymes which operate without coenzymes and cofactors [ 1,2]. Among the functional groups of proteins the imidazole, thiol and carboxyl groups are the most interesting ones with respect to their possible role in enzyme catalysis. While the mechanism of action of these groups in monofunctional catalysis has been thoroughly investigated [3], there exist only few studies on multifunctional cooperative catalysis including imidazole, thiol and carboxyl groups [4-71. We therefore have synthesized a number of peptides containing histidine, cystein and aspartic acid and have looked for possible cooperative effects of the functional groups by an extensive kinetic analysis of their catalytic properties in ester hydrolysis. In the present communication we report the first results of our studies on the catalytic properties of the synthetic peptides.