Erhard Gross

α, β-Unsaturated and Related Amino Acids in Peptides and Proteins

α, β-Unsaturated amino acids are potential precursors for the formation of crosslinkages in peptides and proteins. DEHYDROALANINE and DEHYDROBUTYRINE are constituents of NISIN (from Streptococcus lactis) and SUBTILIN (from Bacillus subtilis). Both peptides are crosslinked via sulfide bridges of no fewer than one residue of lanthionine and four residues of β-methyllanthionine presumably formed by the addition of the sulfhydryl group of cysteine residues across the double bond of dehydroalanine and dehydrobutyrine, respectively. CINNAMYCIN (from Streptomyces cinnamoneus) and DURAMYCIN (from Streptomyces cinnamoneus forma azacoluta) also display the crosslinking features of lanthionine and β-methyllanthionine. The reactive double bond of α, β-unsaturated amino acids is no longer seen in these two peptides. The presence of LYSINOALANINE in cinnamycin and duramycin establishes the imino bridge as a novel type of naturally occurring cross-linkage. The formation of the imino bridge is attributed to the addition of the e-amino group of a lysine residue across the α, βunsaturation of dehydroalanine, a reaction that takes place in nisin, nisin fragments, and subtilin under controlled alkaline conditions. The crypticity of α, β-unsaturated amino acids constitutes a continued impediment to their easy analytical detection. A more broadly based role for α, β-unsaturated amino acids in the physiological environment must not be ruled out at the present time.

The number and nature of α,β-unsaturated amino acids in subtilin

Abstract In subtilin, a peptide produced by Bacillus subtilis , there are present three α,β-unsaturated amino acids, namely, two residues of dehydroalanine and one residue of β-methyldehydroalanine (dehydrobutyrine). Subtilin and nisin, a polypeptide produced by Streptococcus lactis , thus have in common not only the COOH-terminal sequence dehydroalanyllysine but also the number and nature of α,β-unsaturated amino acids.

Some characteristics of the neutrophil receptor for chemotactic peptides

Formylated peptides have been shown to be potent chemoattractants for leukocytes [ 1,2]. With the aid of an intrinsically labelled chemoattractant, f-Nle-Leu-[‘HIPhe (ML[3H]P) [3], a high affinity binding site (Kd 1.5 nM) was demonstrated for formylated peptides on rabbit neutrophils [4]. The no. binding sites/cell was estimated to be -10’. In [S], 0.7 X 10’ sites/cell were estimated. This putative receptor, in addition to binding a number of peptide agonists and antagonists, interacted with a partially purified chemotactic factor produced by Es~h~~hiu cc& 141. Others have demonstrated in human cells a similar receptor for chemotactic peptides [6,7]. Here we present some characteristics of the chemoattractant binding site on the rabbit neutrophil. In an approach similar to that used to characterize other receptors such as the opiate [8] and fl-adrenergic [9] binding sites, we have investigated the correlation between binding and biological activity

The number and nature of α,β-unsaturated amino acids in nisin

Identical COOH-terminal sequences, viz. dehydroalanyllysine, have recently been established for the peptide antibiotics nisin [ 1 ] and subtilin [2] . The release of one equivalent of pyruvyllysine upon treatment of nisin at 100°C for 10 minutes with hydrogen chloride in glacial acetic acid [l] indicated the presence of one residue of the a&unsaturated amino acid. However, carboxymethylcysteine was found in excess of one residue in the hydrolysate of the addition product of mercaptoacetamide to nisin. The excess of carboxymethylcysteine over 1 residue did not result from &elimination of lanthionine. The test for sulfhydryl groups with maleimide was negative; the hydrolysate of the nisin-mercaptan addition product was free of cystine and contained the same number of lanthionine residues as nisin. Oxygen had to be excluded carefully in order to determine the presence of 2 residues of dehydroalanine and 1 residue of P-methyldehydroalanine in the antibiotic.

Agonist–antagonist properties of N-allyl-[D-Ala]2-Met-enkephalin

RECEPTOR displacement of the opiate antagonist naloxone requires concentrations of morphine and other opiate alkaloids similar to those required to elicit analgesia1–3 or inhibit ileal contraction4–5. A new class of opiates with structures based on the discovery of endogenous, morphine-like pentapeptide—enkephalin6—also displace opiate receptor binding with potencies paralleling in vivo activities7–10. Intrinsic activity (agonist or antagonist quality), a different dimension of opiate alkaloid effects in vivo, is closely correlated with the in vitro effects of sodium ion in the incubation medium: while antagonist binding to opiate receptors labelled by radioactive naloxone is almost unaffected by sodium ion addition, opiate alkaloid agonists become 10–60-fold weaker and mixed agonist–antagonists have intermediate downward shifts in apparent affinity after sodium ion addition1,11. We present here an analysis of the sodium sensitivities of the major opiate peptides which have all been identified as ‘agonists’ thus far. In addition, we describe the synthesis, behavioural and in vitro receptor analysis of N-allyl-[D-Ala]2-Met-enkephalin (Fig. 1), a novel peptide whose structure was designed in analogy12 with that of the alkaloid opiate antagonists13 in order to obtain a peptide opiate antagonist, if possible.

The reaction of cyanogen bromide with S-methylcysteine: fragmentation of the peptide 14-29 of bovine pancreatic ribonuclease A.

Summary The S-methylated peptide 14–29 of bovine pancreatic ribonuclease A is cleaved by cyanogen bromide with the formation of the expected fragments in 88% yield based on the conversion of S-methylcysteine.

Antibiotics and peptides with agonist and antagonist chemotactic activity

Abstract It has been found that the polypeptide antibiotics gramicidin S, tyrocidin and bacitracin, containing Leu-Phe or Ile-Phe sequences, are chemoattractants for neutrophils. Related synthetic pentapeptides containing the sequence Leu-Phe have stronger biological activities, provided the N-terminal residue is acylated. The formylated peptide f-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe is a potent agonist (4 × 10 −9 M) whereas the t-butyloxycarbonyl analog is a good antagonist (8 × 10 −7 M).

Synthesis of cyclotetrapeptides, am-toxin analogs, containing α-hydroxyalanine

Abstract Condensation of amide with α-keto acid to yield a α-hydroxyalanine (α-Hyala) or dehydroalanine residue was applied to syntheses of analogs of AM-toxins, cyclotetradepsipeptides. Cyclo (-α-Hyala-L-Ala-L-Val-L-Phe-), cyclo (-α-Hyala-L-Ala-L-Hmb-L-Phe-) and cyclo (-α-Hyala-L-Ala-L-Hmb-L-Tyr-) (Hmb, 2-hydroxy-3-methylbutanoic acid) were obtained from the corresponding pyruvyl-tripeptide amides in good yields by the treatment of anhydrous hydrogen fluoride.

NONENZYMATIC CLEAVAGE OF PEPTIDE BONDS AND MULTIPLE-MOLECULAR FORMS OF ENZYMES

Nonenzymatic cleavage of peptide bonds is a relatively new technique for the fragmentation of peptides and proteins. As is implied by the term “nonenzymatic,” the commonly employed tools of peptide and protein fragmentation-the proteolytic enzymes-are no longer used. They are replaced by simple chemical compounds. Several of these reagents will be discussed later. Prerequisite for the nonenzymatic cleavage of a peptide bond is the presence of a functional group in the side chain of one of the two participating amino acids. Certain techniques for the nonenzymatic cleavage of peptide bonds are highly selective, and the reagent employed will react with one, and only one, of the commonly occurring amino acids. These techniques are applicable to protein fragmentation. Once applied to isozymes, they are capable of producing fragments which are microheterogeneous. I

The Peptide Bond

Publisher Summary This chapter describes the nature and conformation of peptide bond. One of the most fascinating aspects of the unique nature of the peptide bond is the ability of nascent biosynthetic peptide chains to fold spontaneously into the complex three-dimensional structures, which are characteristic of proteins. The chapter presents a solution conformation for oxytocin that revealed the presence of a type II β-turn involving the sequence -Tyr-Ile-Gln-Asn- within the 20-membered cyclic moiety of the hormone. The chain is folded back into an antiparallel pleated sheat conformation with the disulfide bridge closing the ring and stabilizing the structure. The backbone NH of asparagine-5 is hydrogen bonded to the C=O of tyrosine-2 and provides additional intramolecular stabilization. The CONH2-terminal tail moiety forms a second β-turn, comprising residues -Cys-Pro-Leu-Gly-, which folds the tail over one side of the ring and is stabilized by another hydrogen bond between the NH of the leucine-8 and the side chain C=O of the asparagine-5 residues. All peptide bonds are of trans configuration, including the Cys–Pro bond. Similar but not identical conformations have been deduced for lysine-vasopressin, arginine-vasopressin, and arginine-vasotocin.