Development of an internally quenched fluorescent substrate and a continuous fluorimetric assay for Streptococcus pneumoniae signal peptidase I.

Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria and serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. In this paper, we describe a novel fluorogenic substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, in which 2-aminobenzoic acid (Abz) and 3-nitrotyrosine (Y(NO2)) were used as the fluorescent donor and acceptor, respectively. The substrate can be cleaved by both Streptococcus pneumoniae and Escherichia coli SPase I. Upon cleavage of the fluorogenic substrate by SPase I, the fluorescent intensity increases and can be monitored continuously by spectrofluorometer. Kinetic analysis with S. pneumoniae SPase I demonstrated that the K(m) value for the substrate is 118.1 microM, and the k(cat) value is 0.032 s(-1). Mass spectrometric analysis and peptide sequencing of the two cleaved products confirmed that the cleavage occurs specifically at the predicted site. More interestingly, the positively charged lysine in the N-terminus of the substrate was demonstrated to be important for effective cleavage. Phospholipids were found to stimulate the cleavage reaction. This stimulation by phospholipids is dependent upon the N-terminal charge of the substrate, indicating that the interaction of the positively charged substrate with anionic phospholipids is important for maintaining the substrate in certain conformation for cleavage. The substrate and assay described here can be readily automated and utilized for the identification of potential antibacterial agents.

Structure of an inhibitor complex of the proteinase from feline immunodeficiency virus.

The crystal structure of a recombinant form of the proteinase encoded by the feline immunodeficiency virus (FIV PR) has been solved at 2 A resolution and refined to an R-factor of 0.148. The refined structure includes a peptidomimetic, statine-based inhibitor, LP-149, which is an even more potent inhibitor of HIV PR. Kinetic parameters were obtained for the cleavage of five substrates by FIV PR, and inhibition constants were measured for four inhibitors. The structure of FIV PR resembles other related retroviral enzymes although few inhibitors of HIV PR are capable of inhibiting FIV PR. The structure of FIV PR will enhance our knowledge of this class of enzymes, and will direct testing of new proteinase inhibitors in a feline animal model.

Model peptides to study the effects of P2 and P3 substitutions in statine-containing HIV proteinase inhibitors.

Through a series of synthetic model peptides, we have examined the structural requirements of the P2 and P3 residues in statine-based HIV protease (PR) inhibitors. Results agree with the general observations that, the more bulky the P3 aromatic hydrophobic side chain, the more potent is the inhibitor. At P2, an isopropyl side chain is critical in maintaining potency. Three-dimensional modeling demonstrates that the steric bulk of a leucyl residue or the unfavorable energy transfer, from water to enzyme, for a basic amino acid residue at P2 markedly compromises activity. A naphthylalaninyl-valyl P3-P2 substituted analogue inhibits PR with an IC50 value of 6 nM, and was also effective as an antiviral agent.

Minimal sequence requirement of thrombin receptor agonist peptide.

A site-specific proteolytically generated neoamino terminus of the thrombin receptor having a sequence SFLLRNPNDKYEPF- has been reported to be a functional ligand of the receptor. This discovery raises question on the precise structural requirements of the "tethered ligand" responsible for receptor activation and signal transduction. By examining the agonist activity of a panel of synthetic sequence analogues of thrombin receptor agonist peptides (TRAP) on human platelet aggregation, we determined that the minimal sequence of the human platelet thrombin receptor ligand is SFLL-amide (TRAP1-4, EC50 = 300 uM). An extension of TRAP1-4 by an additional Arg-Asn segment yielded the most potent agonist among the series (TRAP1-6, EC50 = 1.3 microM). Based on the structure-activity relationships, we hypothesized a model of the ligand-binding site of the human platelet thrombin receptor that accommodates a hexapeptide structure. TRAP1-6, when administered intravenously, induced marked intravascular platelet aggregation in the anesthetized guinea pigs.

Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation.

Latent human fibroblast collagenase (HFC) can be activated by a variety of seemingly disparate means. In addition to the well-characterized activation by trypsin and organomercurial compounds, the enzyme can be activated to various extents by surfactants such as sodium dodecyl sulfate, by chaotropic ions such as SCN-, by disulfide compounds such as oxidized glutathione, by sulfhydryl alkylating agents such as N-ethylmaleimide, and by oxidants such as NaOCl. The underlying basis for these activations is the modification, exposure, or proteolytic release of the Cys73 residue from its habitat in the latent enzyme where it is thought to be complexed to the active-site zinc atom. This residue is not accessible for reaction with small molar excesses of dithionitrobenzoate in native, latent HFC. However, on addition of EDTA, this residue becomes fully exposed and is quantitatively labeled. All modes of activation of latent HFC are believed to involve the dissociation of Cys73 from the active-site zinc atom and its replacement by water, with the concomitant exposure of the active site. This is thought to be the primary event that precedes the well-known autolytic cleavages that are observed following the appearance of collagenase activity. The dissociation of Cys73 from the zinc atom in the latent enzyme "switches" the role of the zinc from a noncatalytic to a catalytic one. This "cysteine switch" mechanism of regulation may be applicable to the entire collagenase gene family.

Preparation and reconstitution with divalent metal ions of class I and class II Clostridium histolyticum apocollagenases.

Both gamma- and zeta-collagenases from Clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at pH 7.5 in the presence of 10 mM CaCl2 with KI values of 0.11 and 0.040 mM, respectively. The inhibition is caused by removal of the single, active-site Zn(II) present in each of these enzymes. The nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme. Dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis against buffer containing no chelating agent, gives the respective apocollagenases. Both apoenzymes can be instantaneously and fully reactivated by the addition of 1 equiv of Zn(II). Variable amounts of activity are restored to both apocollagenases by Co(II) and Ni(II) and to gamma-apocollagenase by Cu(II). The activity titration curve for gamma-apocollagenase with Co(II) and Scatchard plots for the reconstitution of gamma-apocollagenase with Cu(II) and Ni(II) and of zeta-apocollagenase with Ni(II) and Co(II) indicate that all activity changes are the result of binding of a single equivalent of these divalent metal ions at the active site of the collagenases. Cd(II) and Hg(II) do not restore measurable activity to either apoenzyme.

Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases.

Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange. The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min. However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent. The kinetic parameters Kcat and KM have been determined for each of the active metallospecies. The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM. This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.