Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A.

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [Löbermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.

Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures.

Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. An enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the general base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the N(3) atom of the guanidinium group. OH- 377 adds to the C(1) atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.

Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida.

The three-dimensional crystal structure of creatine amidinohydrolase (creatinase EC 3.5.3.3) from Pseudomonas putida, a dimeric enzyme with a molecular weight of 97,000, has been determined by multiple isomorphous replacement, averaging over the local dyad and restrained crystallographic refinement at 1.9 A with a crystallographic R-value of 17.7%. The asymmetric unit contains a dimer. The two chemically identical subunits consist of 403 residues each. A subunit is built up of two domains, a small N-terminal and a larger C-terminal domain. The small domain has a central seven-stranded beta pleated sheet with short helices on the outside. The large domain forms a six-stranded antiparallel beta half-barrel with helices on the outside. The two domains are connected by a segment that links two helices. The binding site of the competitive inhibitor carbamoyl sarcosine, a close analog of the substrate creatine, is located in the center of the large domain and partly covered by the small domain of the other subunit. The carbamoyl group is tightly co-ordinated to a water molecule, which presumably represents the nucleophile involved in hydrolysis of creatine. A catalytic mechanism is proposed on the basis of this structure.

[Cholecystokinin-pancreozymin synthesis. Synthesis of [28-threonine,31-norleucine]- and [28-threonine,31-leucine]cholecystokinin-pancreozymin-(25-33)-nonapeptide]. / Zur Synthese von Cholecystokinin-Pankreozymin. Darstellung von [28-Threonin,31-Norleucin]- und [28-Threonin,31-Leucin]Cholecystokinin-Pankreozymin-(25-33)-nonapeptid.

The syntheses of two analogues related to the C-terminal nonapeptide amide sequence 25-33 of cholecystokinin-pankreozymin are described. Based on the primary structure of the CCK-PZ-active caerulein and the experiences gained from the methionine replacement with leucine or norleucine in human little gastrin I, the analogues were designed by substituting methionine 28 with threonine, and methionine 31 with leucine and norleucine, respectively. Using a new method for the synthesis of tyrosine-O-sulfate-containing peptides, developed in our laboratory, and applying acid-labile side-chain protection in combination with the benzyloxycarbonyl group, the fully protected nonapeptide amide derivatives Z-Arg(Z2)-Asp(OBut)-Tyr-(SO3Ba1/2)-Thr(But)-Gly-Trp-Leu-Asp(OBut)-Phe-NH2 and Z-Arg(Z2)-Asp(OBut)-Tyr(SO3-Ba1/2)-Thr(But)-Gly-Trp-Nle-Asp(OBut)-Phe-NH2, were obtained. Upon hydrogenolytic and subsequent acidolytic removal of the protecting groups, followed by purification via chromatographic procedures the nonapeptide amides were isolated in satisfactory yields at a high degree of purity. In vivo and in vitro assays showed that a substitution of methionine 31 by norleucine with concomitant replacement of methionine 28 by threonine produced a fully active analogue, whereas for the threonine 28, leucine 31 analogue the pankreozymin-activity was lowered by a factor 10.

[Synthesis of tyrosin O-sulfate-containing peptides (author's transl)]. / Synthese von Tyrosin-O-sulfat-haltigen Peptiden.

A new synthetic route to tyrosine-O-sulfate-containing peptides based on the direct use of N alpha-acyltyrosine-O-sulfate is described and exemplified by the synthesis of the [Thr28,Leu31]cholecystokinin-pankreozymin-(25-33)-nonapeptide.

[Side-reactions in peptide synthesis, III. Synthesis and characterization of Nin-tert-butylated tryptophan derivatives (author's transl)]. / Nebenreaktionen bei Peptidsynthesen, III. Synthese und Charakterisierung von Nin-tert-butylierten Tryptophan-derivaten.

It was possible to elucidate a side reaction which had long been assumed to occur during the acidolytic cleavage of protecting groups based on a ter-butyl moiety, by synthesizing Nin-tert-butyltryptophan in different ways. It was found to be absolutely identical with a "modified tryptophan" which was isolated after the total synthesis of a gastrin analogue; Nin-tert-butyl-tryptophan peptides are formed as the main products of a tert-butylation reaction, the mechanism of which is not very clear yet. The proof for a Nin-tert-butylation of tryptophan was obtained by spectroscopic methods, in particular mass spectrometry and nuclear magnetic resonance spectroscopy.

[Side-reactions in peptide synthesis, IV. Charactertzation of C- and C,N-teri-butylated tryptophan derivatives (author's transl)]. / Nebenreaktionen bei Peptidsynthesen. IV. Charakterisierung von C- und C,N-tert-butylierten Tryptophan-Derivaten.

Besides Nin-tert-butylated tryptophan derivatives, other tert-butyl-tryptophan analoga are also found as a result of side-reactions accurring during the acidolytic cleavage of protecting groups being based on a tert-butyl moiety. C-mono-substitution could be detected as well as di- and tri-substitution. All these tert-butylated tryptophans could be isolated in a pure form from the tert-butylation mixture either free or as derivatives and could unequivocally be identified in particular by means of mass spectrometry and nuclear magnetic resonance spectroscopy.

[Direct tert-butylation of tryptophan: preparation of 2,5,7-tri-tert-butyltryptophan (author's transl)]. / Direkte tert-Butylierung des Tryptophans. Herstellung von 2,5,7-Tri-tert-butyltryptophan.

The tert-butylation of free tryptophan under simulated conditions of acidolytic cleavage of protecting groups being based on a tert-butyl moiety does finally yield the tri-substituted amino acid as a main product, namely 2,5,7-tri-tert-butyltryptophan. The structural elucidation of this substance and other tert-butylated products, which were isolated mostly in a pure form, was achieved by means of spectroscopic methods, in particular mass spectrometry and nuclear magnetic resonance spectroscopy.

[Synthesis of human [11-leucine]minigastrin I--II. Purification of the synthetic tridecapeptide amide and isolation of a side-product (author's transl)]. / Zur Synthese von [11-Leucin]Human-Minigastrin I II. Mitteilung: Reindarstellung des synthetischen Tridekapeptidamids sowie Isolierung und Strukturaufklärung eines Synthese-Nebenproduktes.

The preparation of the pure 11-leucine analogue of human minigastrin I, a linear tridecapeptidaemide with full "gastrin activity", from the crude synthetic material obtained after deblocking of the overall protected tridecapeptide amide derivative by means of trifluoroacetic acid, is achieved by ion-exchange chromatography on DEAE-Sephadex A-25 and subsequent partition chromatography on Sephadex G-25. The isolation of a synthetic side-product and its identification as human [10-(Nin-tert-butyl-tryptophan),11-leucine]minigastrin I by means of spectroscopic (UV, fluorescence, 1H-NMR, MS), enzymatic and chromatographic methods is described. The pure tridecapeptide amides human [Leu11]minigastrin I and human [Trp(1'-But)10,Leu11]minigastrin I are isolated in overall yields of 37.4% and 4.6%, respectively (mol-% with regard to the overall protected tridecapeptide amide derivative).

[Total synthesis of human big gastrin I and the 32-leucine analogue (author's transl)]. / Zur Totalsynthese des Human-Big-Gastrins I und seines 32-Leucin-Analogons.

Synthetic Peptide Hormones, Human Big Gastrin I. A new total synthesis of the tetratriacontapeptide amide corresponding to the proposed primary structure of human big gastrin I is described. The synthetic route was based on the preparation of six suitably protected fragments, related to sequence 28-34, 23-27, 21-22, 15-20, 9-14, and 1-8, to be used as building blocks for the total synthesis. The protecting groups were selected according to the Schwyzer-Wünsch strategy of maximum side chain protection based on tertiary alcohols, also for the imidazol function of histidine. Subsequent assembly of the six fragments by three different pathways using the highly efficient Wünsch-Weygand condensation procedure to ensure minimum racemization, followed by deprotection of the synthetic products via exposure to trifluoroacetic acid and final purification by ion-exchange chromatography on DEAE-Sephadex A-25 and partition chromatography on Sephadex G-25, led to human big gastrin I, homogeneous within the limits of the analytical methods used. The biological activity of the synthetic product proved to be 50 percent higher than that of human little gastrin I. The 32-leucine analogue of human big gastrin I was prepared in the same way.

[The synthesis of motilin, III: Purification and characterization of (13-norleucine) motilin and (13-leucine) motilin (author's transl)]. / Zur Synthese von Motilin, III[1]. Reindarstellung und Charakterisierung von [13-Norleucin] motilin und [13-Leucin]motilin

The preparation of the pure docosapeptide H-Phe-Val-Pro-I1e-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Nle-Glu-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln-OH ([Nle13]motilin) and of the analogous [Leu13]motilin from the crude synthetic materials obtained after deblocking of the overall protected docosapeptide derivatives by means of trifluoroacetic acid is described. In a preliminary experiment the separation of crude [Nle13]-motilin into several components, some of which being indistinguishable by thin layer chromatography, was achieved by repeated ion-exchange chromatography on QAE-Sephadex A-25 and SP-Sephadex C-25. Subsequent characterization of some of the isolated side-products, using amino acid analysis as well as spectroscopic (UV, CD) and enzymatic methods, in comparison to the major product enabled conclusions on the reasons of their formation. The undesired formation of des-(Thr6 -Tyr7 Gly8)[Nle13] motilin and of [D-Phe5-Nle13]motilin could be avoided during the subsequent major synthesis of [Nle13] motilin and during the preparation of [Leu13] motilin by changing certain synthetic conditions and, respectively, by using a specially purified protected fragment of the sequence 1-8, being free of diastereomers. Single ion-exchange chromatography on SP-Sephadex C-25 was sufficient for the purification of the so obtained crude synthetic end-products.