High-Throughput Screening of Enzyme Inhibitors: Automatic Determination of Tight-Binding Inhibition Constants

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 Å for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 Å for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.

Section: Expression Of Plasma Kallikrein Protease Domain In P Pastormentioning

confidence: 99%

Expression, Crystallization, and Three-dimensional Structure of the Catalytic Domain of Human Plasma Kallikrein

Tang¹,

Yu²,

Williams³

et al. 2005

“…The concentration of the SBzl substrate was also determined using total hydrolysis with trypsin; the released SBzl moiety was monitored spectrophotometrically at 324 nm in the presence of 0.5 mM 4,4Ј-dithiodipyridine, and concentration was determined using the extinction coefficient of 19,800 M Ϫ1 cm Ϫ1 for the SBzl-thiopyridine conjugate. Active site titration for purified CF40-Gly-NS3pro185 proteases was performed by inhibition with freshly reconstituted aprotinin (18,19). For kinetic studies, CF40-Gly-NS3pro185 proteases were incubated with various concentrations of individual ACMC, AMC, or SBzl peptide substrates at 37°C.…”

Section: Expression and Purification Of Den 1-4 Cf40-gly-ns3pro185-mentioning

confidence: 99%

Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries

Lim

Beer

et al. 2005

232

301

Regulated proteolysis by the two-component NS2B/ NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1 and P3. In contrast, the P2 and P4 substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.Dengue virus is the etiologic agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is the most prevalent arthropod-transmitted infectious disease in humans. Dengue consists of four closely related but antigenically distinct viral serotypes (DEN1-4), 1 of the genus Flavivirus (1, 2).Following primary infection, lifelong immunity develops that prevents repeated assault by the same serotype but does not provide protection from a virus of a different serotype (3). Dengue diseases are endemic in the tropics and subtropics, and the viruses are maintained in a cycle that involves humans and the Aedes aegypti mosquito. Infection with dengue viruses produces a spectrum of clinical illness ranging from a nonspecific viral syndrome to severe and fatal hemorrhagic disease (1, 2). Currently there is no antiviral drug or vaccine available against dengue viruses, and the pathogenesis of the disease is poorly understood.As with other members of the Flaviviridae family, the genomes of the dengue viruses consist of a positive singlestranded RNA of ϳ10,700 bases in length (4). Co-translational processing and post-translational processing of the polyprotein give rise to three structural proteins and at least seven nonstructural proteins (4). The correct processing of these proteins is essential for virus replication and requires host proteases such as signalase and furin (5) and a two-component viral protease, NS2B/NS3 (4). Previous studies have shown that the N-terminal part of NS3 contains trypsin-like protease domain (6) and that the activity of NS3 was dependent on at least 40 amino ...

“…To determine the initial reaction velocities, the program fits reaction progress curves either to a straight line or to the quadratic parabola using an automatic model selection procedure, based on the sequential modified givens orthogonal transformation method (43). In the second stage of the analysis, the program uses an automatic algorithm described recently (44) to fit the initial velocities to the Morrison equation (Equation 1) to determine the apparent inhibition constants K i *.…”

Section: Figmentioning

confidence: 99%

A Broad Spectrum Kunitz Type Serine Protease Inhibitor Secreted by the Hookworm Ancylostoma ceylanicum

Milstone

Harrison

Bungiro

et al. 2000

Although blood-feeding hookworms infect over a billion people worldwide, little is known about the molecular mechanisms through which these parasitic nematodes cause gastrointestinal hemorrhage and iron deficiency anemia. A cDNA corresponding to a secreted Kunitz type serine protease inhibitor has been cloned from adult Ancylostoma ceylanicum hookworm RNA. The translated sequence of the A. ceylanicum Kunitz type inhibitor 1 (AceKI-1) cDNA predicts a 16-amino acid secretory signal sequence, followed by a 68-amino acid mature protein with a molecular mass of 7889 daltons. Recombinant protein (rAceKI-1) was purified from induced lysates of Escherichia coli transformed with the rAceKI-1/pET 28a plasmid, and in vitro studies demonstrate that rAceKI-1 is a tight binding inhibitor of the serine proteases chymotrypsin, pancreatic elastase, neutrophil elastase, and trypsin. AceKI-1 inhibitory activity is present in soluble protein extracts and excretory/secretory products of adult hookworms but not the infective third stage larvae. The native AceKI-1 inhibitor has been purified to homogeneity from soluble extracts of adult A. ceylanicum using size exclusion and reverse-phase high pressure liquid chromatography. As a potent inhibitor of mammalian intestinal proteases, AceKI-1 may play a role in parasite survival and the pathogenesis of hookworm anemia.