Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition

Apel

Molecular and Cellular Biology

1995

The NADPH:protochlorophyllide oxidoreductase precursor protein (pPorA) of barley (Hordeum vulgare L. cv. Carina), synthesized from a full-length cDNA clone by coupling in vitro transcription and translation, is a catalytically active protein. It converts protochlorophyllide to chlorophyllide in a light-and NADPH-dependent manner. At least the pigment product of catalysis remains tightly bound to the precursor protein. The chlorophyllide-pPorA complex differs markedly from the protochlorophyllide-pPorA complex with respect to sensitivity to attack by a light-induced, nucleus-encoded, and energy-dependent protease activity of barley plastids. The pPorA-chlorophyllide complex is rapidly degraded, in contrast to pPorA-protochlorophyllide complexes containing or lacking NADPH, which are both resistant to protease treatment. Unexpectedly, pPorA devoid of its substrates or products was less sensitive to proteolysis than the pPorA-chlorophyllide complex, suggesting that both substrate binding and product formation during catalysis had caused differential changes in protein conformation.

Section: Discussionmentioning

confidence: 98%

A Light-Induced Protease from Barley Plastids Degrades NADPH:Protochlorophyllide Oxidoreductase Complexed with Chlorophyllide

Apel

Molecular and Cellular Biology

1995

Journal of Biological Chemistry

“…Well defined S4, S3, S2, S1, S1Ј, S2Ј, S3Ј, and S4Ј subsite pockets for the amino acid side chains of the substrate (5) are additional hallmarks of these enzymes. A peptide analogue of microbial origin, pepstatin, is the definitive, general inhibitor of aspartic proteases (7). In general, human cathepsin D is specific for hydrophobic patches in proteins, with an ostensibly anomalous, additional preference for glutamate in the P2 position (8 -11).…”

mentioning

confidence: 99%

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Brinkworth¹,

Prociv²,

Loukas³

et al. 2001

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4 were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH 2 ]Leu-Tyr-Tyr-Ser-NH 2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4 residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.Blood flukes, hookworms, and the malaria parasites are among the most important pathogens of humans in terms of both numbers of people infected and the consequent morbidity and mortality (1). Although phylogenetically unrelated, these parasites all share the same food source; they are obligate blood feeders, or hematophages. Hb from ingested or parasitized erythrocytes is their major source of exogenous amino acids for growth, development, and reproduction; the Hb, a ϳ64-kDa tetrameric polypeptide, is comprehensively catabolized by parasite enzymes to free amino acids or small peptides. Intriguingly, all these parasites appear to employ cathepsin D-like aspartic proteases to make initial or early cleavages in the Hb substrate (2-4).The vertebrate endopeptidase, cathepsin D (EC 3.4.23.5), is a member of the aspartic protease category of hydrolases, which also includes renin, pepsin, chymosin, cathepsin E, HIV 1 protease, and several other enzymes (5, 6). Cathepsin D is expressed in a diverse range of mammalian cells and tissues and is located predominantly in lysosomes (6). The molecule comprises two rather similar lobes, each incorporating a homologous Asp-Thr-Gly catalytic site motif, with the substrate binding groove located between these lobes. In aspartic proteases generally, the nucleophile that attacks...

Proc. Natl. Acad. Sci. U.S.A.

“…13) has the weakest K i at 80 M (28), whereas the strongest binding ligand is pepstatin A (no. 17), with a K i of 45 pm (29). These values give an estimation of the K i 's needed for determination of candidates as binding ligands by using the IEMS assay.…”

Section: Resultsmentioning

confidence: 99%

Mass spectrometry and immobilized enzymes for the screening of inhibitor libraries

Cancilla

Leavell

Chow

et al. 2000

A technique has been developed to rapidly screen enzyme inhibitor candidates from complex mixtures, such as those created by combinatorial synthesis. Inhibitor libraries are screened by using immobilized enzyme technologies and electrospray ionization ion cyclotron resonance mass spectrometry. The library mixture is first sprayed into the mass spectrometer, and compounds are identified. The library is subsequently incubated with the immobilized enzyme of interest under the correct conditions (buffer, pH, temperature) by using an excess of enzyme to ensure a surplus of sites for ligand binding. The immobilized enzyme͞inhibitor mixture is centrifuged, and an aliquot of supernatant is again analyzed by electrospray ionization mass spectrometry. Potential inhibitors are quickly identified by comparison of the spectra before and after incubation with the immobilized enzyme. Non-inhibitors show no change in ion intensity after incubation, whereas weak inhibitors exhibit a visible decrease in ion abundance. Once inhibitor candidates have been identified, the library is reinjected into the mass spectrometer, and tandem mass spectrometry is used to determine the structure of the inhibitor candidates as needed. This method has been successfully demonstrated by identifying inhibitors of the enzymes pepsin and glutathione S-transferase from a 19-and 17-component library, respectively. It is further shown that the immobilized enzyme can be recycled and reused for continuous screening of additional new libraries without adding additional enzyme.D uring the last decade new combinatorial techniques have been developed that allow for synthesis of vast quantities of potential therapeutic compounds (1-4). Although remarkable advances in generating complex libraries of molecules have been made, the analytical process of analyzing and screening the immense numbers of compounds generated is currently lacking. To keep pace with the synthetic process, contemporary analytical techniques must be capable of screening a large number of compounds in a high-throughput manner with precision and accuracy.Notable progress has been made in overcoming the problem of screening extensive libraries that are created by combinatorial methods using MS (5-8). At present, a variety of front-end affinity-selection techniques are used in conjunction with MS to determine potentially active compounds. Although the nomenclature of the techniques may be different, the solution phase screening methods are all generally based on the same principles. The screening process is initiated by forming a protein͞ligand complex, followed by isolation of the complex by using, for example, size exclusion chromatography (9-12) or a molecular weight cut-off membrane (13,14). Determination of bound ligands requires dissociation of the complex, followed by chromatographic-mass spectrometric detection. Some examples include pulsed ultrafiltration MS, developed by van Breemen and coworkers, as an elegant combinatorial library screening methodology (13, 15). There are also a vari...