Thesis (M.S.)--University of Wisconsin--Madison, 1980. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 64-66).

The active site of porcine microsomal aminopeptidase was probed by studying the inhibition of the enzyme using derivatives of ethylenediamine and diaminopropionic acid. In addition, some amino acids, substituted hydroxamates and phosphates were also tested. In order to synthesize diaminopropionic acid derivatives, CBZ-amino acid p-nitrophenyl esters were reduced to the corresponding aldehydes by lithium tri-t-butoxy-aluminohydride. Through the Strecker synthesis, the aldehyde intermediates were converted to diaminopropionitrile analogues which were then hydrolysed in acid to the desired products. Unfortunately, these compounds were not potent inhibitors for this enzyme. α-Amino acids were found to be better inhibitors than their β-amino counterparts and the Kᵢ of α-leucine was about 7-fold lower than its B-analogue. The amino group position of the amino acids is therefore important for enzyme recognition. On the other hand, N-alkylation of ethylenediamine was observed to abolish its inhibition potential. Furthermore, another unexpected finding in this work is that N- or 0-methylation of the hydroxamate group hinders the ability of these inhibitors to act as a bidentate zinc ligand. Although some phosphate derivatives that we tested showed poor inhibitory potency, phosphonamidate, a potential transition state analogue, might serve as a powerful inhibitor. In summary, the relationship between the structure and inhibitory potency of some inhibitors was demonstrated. / Thesis / Master of Science (MSc)

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The pathway representing methionine integration and excision is an increasingly important target in drug design. Methionine aminopeptidase (MetAP), a metalloprotease responsible for cleaving the N-terminal methionine from nascent peptides, has been the object of many studies aimed to produce potential anti-bacterial, anti-fungal and anti-angiogenic agents. Though clinical trials are underway for several of these compounds, like fumagillin and CKD-731, they are still flawed based on their relatively weak inhibition and their physiological side effects. Therefore, the search for novel and potent inhibitors continues. Previous work has utilized phosphinic and phosphonic acid derivatives of methionine in co-crystallization studies with Escherichia coli MetAP (eMetAP). The aim of the research presented in this work is to study and assay various phosphorus- and sulfur-containing compounds as inhibitors and substrates in an effort to learn more about the biochemical machinery underlying MetAP catalysis. As well, we outline a predictive molecular modeling approach to MetAP inhibitor design to assist in identifying lead candidates amongst a body of possible molecular inhibitors. Ultimately, we not only hope to have identified key functional properties of molecules potentially useful as MetAP inhibitors, but also to have contributed to the knowledge base of the mechanistic features involved in this enzyme’s catalysis.

The pathway representing methionine integration and excision is an increasingly important target in drug design. Methionine aminopeptidase (MetAP), a metalloprotease responsible for cleaving the N-terminal methionine from nascent peptides, has been the object of many studies aimed to produce potential anti-bacterial, anti-fungal and anti-angiogenic agents. Though clinical trials are underway for several of these compounds, like fumagillin and CKD-731, they are still flawed based on their relatively weak inhibition and their physiological side effects. Therefore, the search for novel and potent inhibitors continues. Previous work has utilized phosphinic and phosphonic acid derivatives of methionine in co-crystallization studies with Escherichia coli MetAP (eMetAP). The aim of the research presented in this work is to study and assay various phosphorus- and sulfur-containing compounds as inhibitors and substrates in an effort to learn more about the biochemical machinery underlying MetAP catalysis. As well, we outline a predictive molecular modeling approach to MetAP inhibitor design to assist in identifying lead candidates amongst a body of possible molecular inhibitors. Ultimately, we not only hope to have identified key functional properties of molecules potentially useful as MetAP inhibitors, but also to have contributed to the knowledge base of the mechanistic features involved in this enzyme’s catalysis.

Unlike most other host-specific mycoplasmas, Mycoplasma gallinarum exists as a commensal with a host range including most poultry as well as some mammals. This property of M. gallinarum may reflect unique mechanisms for its colonization and persistence in hosts. Whereas M. gallinarum shows leucine and arginine aminopeptidase activity, the genes encoding the enzymes had not been cloned and characterized. We identified an aminopeptidase gene (APN) by oligonucleotide hybridization to a genomic library of M. gallinarum in lambda ZAPII bacteriophage. Nucleotide sequence analysis of overlapping phage clones identified a 1,362 bp open reading frame (ORF) encoding a putative leucine aminopeptidase gene. Database searches indicate that this ORF has 68% nucleotide identity and 51% amino acid identity with the M. salivarium leucine aminopeptidase gene. The active sites of the leucine aminopeptidases in other eukaryotes and prokaryotes were conserved in the cloned aminopeptidase gene. Northern-blot hybridization analysis showed that this ORF is expressed as a 1.5 kb transcript. Southern-blot hybridization analysis demonstrated this gene was present as a single copy in M. gallinarum. Characterization of the leucine aminopeptidase demonstrated that it is a metallo-aminopeptidase (EC 3.4.11.1) and is located in the cytoplasm with a weak interaction with the cell membrane. The subcellular location was further confirmed by immunoblotting with polyclonal anti-recombinant APN serum and M. gallinarum Triton-114 partitions. Immunoblotting results with sera from three chickens experimentally infected with M. gallinarum showed that there were very few proteins in M. gallinarum exposed to the host immune responses and that leucine aminopeptidase was not able to stimulate production of specific humoral antibody. Our results suggest that this leucine aminopeptidase play a role in nutrition supply for the host adaptation of M. gallinarum and that the enzyme was not strongly immunogenic.

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The active site of porcine kidney microsomal aminopeptidase was investigated using single, multiple and EDTA inactivation kinetic studies. Good inhibitors invariably contained a zinc-coordinating group such as the mercapto moiety, which proved to be the best ligand for aminopeptidase. Due to the potency of β -mercaptoethylamine, derivatives of this compound were examined for aminopeptidase inhibition. (S)-2-amino-4-methyl-l-pentanethiol (L-leucinthiol) exhibited the largest potency and specificity towards aminopeptidase when compared against carboxypeptidase A and thermolysin, two similar zinc-peptidases. The presence of a zinc-coordination subsite, two hydrophobic pocket subsites and a second amine-binding subsite (distinct from that responsible for substrate recognition) were discerned and the binding modes of amino acid hydroxamates and mercaptoamines compared using Yonetani-Theorell inhibition kinetics. Aminopeptidase does not show virtually any stereoselectivity between L-and D-leucine hydroxamate while greater than a 1,000-fold preference is seen for L-leucinthiol over the D isomer. Also, the amino group of mercaptoamines is crucial to the binding of these inhibitors whereas that of the hydroxamate compounds does not seen to contribute much to their binding. The differences in binding between hydroxamates and mercaptoamines are postulated to be a consequence of the product analogue nature of the former and transition state analogue character of the latter. L-leucine hydroxamate is proposed to bind in a backwards orientation while the D isomer binds in the normal substrate-like position. Similarly, L-leucinthiol is proposed to bind in the same fashion as substrate. Design of future inhibitors should endeavour to: (1) lower the pᵏₐ of the α-amino group, (2) include an extended chain structure capable of binding to additional hydrophobic pockets, (3) incorporate a second amine moiety into the structure to interact with the second amine-binding subsite and (4) replace the mercapto group with a more potent zinc ligand such as the selenol group. / Thesis / Master of Science (MSc)