H-Ala-Ala-pNA

Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from pH 4 to 12 for 1 h with a t(1/2) of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, D: -Val-Leu-Lys-pNA.

This review documents my research for the past 45 years in peptide chemistry. Initially, in order to study the structure-activity relationships of active center of alpha- and beta-melanocyte stimulating hormones (H-His-Phe-Arg-Trp-Gly-OH), we employed D-amino acids. That approach yielded first published report in 1965 of antagonists containing D-amino acids. Monkey beta-melanocyte stimulating hormone (beta-MSH), an 18 amino acid peptide stimulated pigment cells. We synthesized beta-MSH and fragments thereof, and studied in detail structure-activity relationships. A major and valuable result revealed that the C-terminal pentadecapeptide of beta-MSH exhibited higher MSH activity than the parent hormone providing a new question; namely, what was the role of the N-terminal tripeptide? In order to identify the novel enzyme, spleen fibrinolytic proteinase (SFP), I developed a specific chromogenic substrate, Suc-Ala-Tyr-Leu-Val-pNA, and a specific inhibitor, Suc-Tyr-D-Leu-D-Val-pNA, once again employing my D-amino acid strategy. SFP was purified by affinity chromatography using Suc-Tyr-D-Leu-D-Val-pNA as the bound ligand. The success of this approach provided me the incentive to develop a variety of potential drugs. Thus, I prepared a specific plasmin inhibitor (YO-2) and a plasma kallikrein inhibitor (PKSI-527). Next, my research developed novel opioid receptor specific opioid agonists and antagonists based on 2',6'-dimethyl-L-tyrosine (Dmt) dimers coupled with unique pyrazinone ring as a spacer. They exhibited potent oral antinociceptive activity acting through the mu-opioid receptor. Potent mu-receptor agonists (H-Dmt-Pro-Phe/Trp- Phe-NH(2)) were transformed into highly selective mu-receptor antagonists (N-allyl-Dmt-Pro-Phe/Trp-Phe-NH(2)), which reversed ethanol-induced increases in GABAergic neurotransmission, suggesting the possibility that they may emerge as candidates for the treatment of ethanol addiction.

Tandem mass spectrometric experiments have been carried out on the protonated amides H-Gly-Ala-NH2, H-Ala-Gly-NH2, H-Ala-Val-NH2, H-Val-Ala-pNA, H-Leu-Phe-NH2, H-Phe-Leu-NH2, H-Phe-Tyr-NH2 and H-Tyr-Phe-NH2 with particular emphasis on the fragmentation of the isomeric a2 ions derived therefrom. Primary fragmentation reactions of the protonated amides involve formation of the y1" and b2 ions with further fragmentation of the b2 ion to form the a2 ion which fragments to form iminium ions. Collision-induced dissociation studies of the mass-selected a2 ions were carried out. For the Gly-Ala, Ala-Gly and Val-Ala a2 ions, weak signals were observed corresponding to loss of CO from the a2 ion. With the exception of the Gly-Ala, Ala-Gly and Val-Ala a2 ions, both possible iminium ions (a1 and the internal iminium ion) are observed with the most abundant being that formed by proton attachment to the imine of higher proton affinity. The results provide strong support for the recently proposed (El Aribi et al. J. Am. Chem. Soc. 2003; 125: 9229) mechanism of fragmentation of a2 ions which involves elimination of CO from the a2 ion to form a proton-bound complex of two imines. Based on this mechanism ab initio calculations of the total energies of the a2 ions and the transition states for fragmentation have been carried out giving the energy barrier for fragmentation of each a2 ion. The experimental results are interpreted in terms of these energetics data, unimolecular rate constants calculated by using the RRKM theory, and the imine proton affinities.