SynopsisA theoretical analysis of the conformation of the octapeptide hormone Am1, Val5 angiotensin I1 has been carried out by semiempirical potential energy calculations. A preliminary study of the Alas-Pro-Ala molecule, which mimics the angiotensin backbone, provided us with likely backbone structures on which the effect of the full side chains of the hormone could be assessed. For angiotensin 11, the calculations show that only a small number of folded, compact conformations have a high probability of existence. This is the consequence of favorable packing and of the presence of proline in position 7. These results are consistent with various experimental data, both structural and biological. This method is readily applicable to the study of analogs of the hormone or to other peptides of comparable size.

No abstract

An immunological approach was used for the study of ornithine carbamoyltransferase (OTCase) evolution in bacteria. Antisera were prepared against the anabolic and catabolic OTCases of Pseudomonas aeruginosa and Aeromonas formicans as well as against OTCase and putrescine carbamoyltransferases from Streptococcus faecalis; these antisera were then tested against the unpurified OTCases, either anabolic or catabolic, of 34 bacterial strains. Extensive crossreactions were observed between the antisera to catabolic OTCases from P . aeruginosa, A . formicans and S . faecalis and the catabolic enzymes from other species or genera. These antisera cross-reacted also with the anabolic OTCases of strains of the Enterobacteriaceae but not with the anabolic OTCases of the same species or of other species or genera. The cross-reaction measured between the antisera against P. aeruginosa anabolic OTCase and the anabolic OTCases of other Pseudomonas were largely in agreement with the phylogenic subdivision of Pseudomonas proposed by N. J. Palleroni. The correlation was also significantly higher with the anabolic enzyme of an archaeobacterium, Methanobacterium thermoaceticum, than with the catabolic or anabolic OTCases from other genera in the eubacterial line. The antiserum raised against A . formicans anabolic OTCase was quite specific for its antigen and appeared to be raised against the heaviest of the various oligomeric structures of the enzyme. INTRODUCTIONAnabolic ornithine carbamoyltransferase (OTCase; EC 2.1 .3.3) functions in the biosynthesis of arginine and catalyses the formation of citrulline and phosphate from ornithine and carbamoylphosphate. The anabolic OTCase from a variety of organisms occurs as a trimeric molecule with a subunit molecular mass ranging from 35 to 40 kDa. This trimeric structure also exists in other enzymes involved in carbamoylphosphate metabolism, namely in aspartate carbamoyltransferase (ATCase; EC 2.1 .3.2) and in putrescine carbamoyltransferase (PTCase; EC 2.1.3.6) (Vickers, 1981;Cunin et al., 1986).Catabolic OTCase is part of the catabolic arginine deiminase pathway and catalyses the formation of carbamoylphosphate and ornithine from citrulline and phosphate. Catabolic OTCases are usually large proteins composed of three, six, eight, nine or more identical subunits (Legrain et al., 1977;Marshall & Cohen, 1972;Baur et al., 1987).Arginine prototrophic organisms that possess the arginine deiminase pathway have two OTCases, one anabolic and the other catabolic. In Pseudomonas putida and Pseudomonas aeruginosa, both enzymes function unidirectionally. The Pseudomonas catabolic enzymes display intense allosteric behaviour which probably results from their large oligomeric structure, , 1987). In this context it is not clear whether trimeric and multimeric OTCases are similar proteins resulting from convergence, or homologous proteins resulting from the differentiation and specialization of an ancestral carbamoyltransferase.To answer this question we have determined the amino acid sequence of the t...

The tripeptide Nα,Nɛ‐diacetyl‐l‐lysyl‐d‐alanyl‐d‐alanine (Ac2‐ l‐LLys1‐dAIa2‐dAIa3), which is the standard substrate of the Zn2+ G and serine R61 d‐alanyl‐d‐alanine peptidases, and several ldd tripeptide analogues where the size and/or the electrical charge of the side chains at position 1, 2 or 3 have been modified (alterations affecting more than one position at the same time were not investigated) have been submitted to conformational analyses based on both short‐range and long‐range interactions. Among the many backbone conformers of minimal energy of the øii space that have been characterized, four types of conformers are the most probable ones. Depending on the peptides, these conformers may have varying relative probability P values so that the leader conformer is not always the same, but, in all cases, the sum of their P values is 90% or more. With the Gly1, Gly2 or Gly3 analogues (which encompass a larger conformational space), the above ∑P values are still as high as 35–50%. All the above tripeptides bind to the serine d‐alanyl‐d‐alanine peptidase and with the exception of the Gly3 and Gly2 analogues, to the Zn2+d‐alanyl‐d‐alanine peptidase with virtually the same efficacy, at least within a range of variation of the Km values for the substrates or the Ki values for the inhibitors, which is less than one order of magnitude. Structural variations at position 1, 2 or 3 in the peptides that are compatible with efficient binding are not necessarily compatible with substrate activity, thus converting the modified peptides into competitive inhibitors. In particular, substrate activity requires a long side chain at position 1 in the peptides. Conformational analyses of Ac2‐lLys‐dAla‐dAla show that the main backbone has a tendency to adopt a ring‐like shape from which the lLYS side chain protrudes as an extended structure. This latter structure forms with the C‐terminal d‐alanyl‐d‐alanine an angle varying between 120° and 180° (depending on the conformers) so that its N‐terminal acetyl group is about 1–1.5 nm apart from the scissile amide bond. High turnover numbers (at enzyme saturation) also require a dAla at position 2 with both d‐alanyl‐d‐alanine peptidases and at position 3 in the case of the serine d‐alanyl‐d‐alanine peptidase. Finally, all the conformers of the lAla2 and lAla3 analogues of Ac2‐lLys‐dAla‐dAla fall outside the backbone conformational space that comprises the φiφi angles exhibited by the four types of conformers of the ldd tripeptides. The lAla2 and lAla3 tripeptide analogues do not bind to the serine d‐alanyl‐d‐alanine peptidase (at least at a 10 mM concentration) but they behave as noncompetitive inhibitors of the Zn2+d‐alanyl‐d‐alanine peptidase.

Conformational energy calculations were carried out on H-Pro-Leu-Gly-NH2, the factor that inhibits the release of melanocyte stimulating hormone, and its biologically active analog, H-Pro-Ala-Gly-NH2.Both peptides were found to be relatively compact molecules that retain, however, some degree of flexibility. After structure refinement, H-Pro-Leu-Gly-NH2 possesses at least three preferred compact conformations. Two of these conformations occupy rather broad and flat energy troughs, while a third occupies a narrow and deep potential energy well. This third structure, which consists of a 10-membered S-turn closed by a (4 -* 1) hydrogen bond between the proton of the trans carboxamide of Gly and the C=O of Pro, is the one that was proposed for H-ProLeu-Gly-NH2 in dimethylsulfoxide and was also found by x-ray analysis.