Ivo Bláha

Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B

The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.

Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame

Protein p6 from HIV-1 gag open reading frame is reported to affect both the final phase of assembly of the viral particle and the early stage of the gag polyprotein maturation in vitro. Two separate hypotheses have been proposed, on only one of these reported effects. We think that both observations may be eventually explained if p6 protein strongly inhibits the HIV-1 proteinase. Protein p6 was synthesised by solid-phase peptide synthesis. Several methods of folding the p6 protein were tested, each resulting in the random structure according to both CD and 1D proton NMR spectra. A uniformly high exposure of NH protons to the solution was confirmed by temperature-dependent NMR spectra and isotope exchange experiments. Thus the p6 protein does not have any rigid conformation in solution. A rigid structure is not formed after further cleavage by HIV-1 proteinase as neither the protein nor its fragments are cleaved by this proteinase. In addition, the p6 protein itself does not act as inhibitor of HIV-1 proteinase. This excludes a direct role of p6 protein and supports the hypothesis that p6 is involved in forming the appropriate structure of gag polyprotein precursor. The role of slowly cleaved tight gag-proteinase in the final stage of maturation may be to slow down maturation of the precursor polyproteins prior to their transport to final location in the membrane.

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild‐type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV‐1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV‐1 PR. This dramatic alteration of MAV PR activity achieved by site‐directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV‐1 proteinases.

Subsite specificity of the proteinase from myeloblastosis associated virus

The subsite requirements of the aspartic proteinase from the mycloblastosis‐associated virus (MAV) for the cleavage of peptide substrates were studied with a series of synthetic peptides of general structure Ala‐Thr‐P4‐P3‐P2‐P1 ★ Nph‐Val‐Arg‐Lys‐Ala. The residues in positions P4, P3, P2 and P1 were varied and the kinetic parameters for the cleavage of substrates in 2.0 M NaCl were spectrophotometrically determined at pH 6.0 and 37°C. The acceptance of amino acid residues in particular subsites is similar to that observed with the human immunodeficiency virus type 1 (HIV‐1) proteinase in our earlier studies on the same substrate series: hydrophobic or aromatic residues are preferable in P1 position, a broad variety of residues are acceptable in P3 whereas the residues occupying P2 plays the decisive role in the substrate cleavage as evidenced by its dramatic influence on both k cat and K m values. The most remarkable difference between the two enzymes was found in P3 and P4 subsites. In P3, the introduction of negatively charged glutamate increases the substrate binding by the MAV proteinase 12‐fold and decreases binding by the HIV‐1 proteinase. In P4, Pro in this series is a favourable residue for the MAV proteinase and is strongly inacceptable for HIV‐1 the proteinase. The pH profile of the cleavage was studied with a chromogenic substrate and differences between HIV‐1 and MAV proteinases are discussed.

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self‐processed proteinase. Purification to homogeneity was achieved by ion‐exchange chromatography and reverse‐phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

Protein-engineered proteinase of myeloblastosis associated virus, an enzyme of high activity and HIV-1 proteinase-like specificity.

All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases, are of similar size and of homologous primary structure; they all act catalytically in the form of highly symmetric molecular dimers.1 Detailed studies of retroviral proteinases were carried out on two almost identical proteinases of MAV2,3 and RS V4 (representing the group of avian retroviruses) and on the HIV proteinase.5,6 The knowledge of the 3D structure,2,4,5 catalytic activity and substrate specifity3,6 of the MAV and the HIV proteinase has changed the notion of their general similarity since several features that distinguish each proteinase from the other were revealed. The HIV-1 proteinase has a considerably higher activity3,6 which reflects the different conditions of the expression and action of this enzyme in vivo: 7 The “coding strategy” of MAV allows the expression of the proteinase from the first (gag) open reading frame and provides for the high (i.e. stoichiometrical) level of the relatively “weak” enzyme whereas the smaller amount of the more active HIV enzyme is a result of infrequent translational frameshift events that occur in the overlapping region of the gag and pol reading frames.8 The substrate specificities of retroviral proteinases seem complex and the requirement for a side chain in an individual subsite of a substrate is an outcome of the combination of residues occupying other closely located subsites.3 The two proteinases (MAV and HIV) show rather promiscuous substrate specificity, nevertheless several differences can be traced. We made an attempt to use protein engineering of the MAV proteinase to tackle directly problems of structural basis of these differences and, vice versa, to make more precise conclusions on the functional importance of the individual elements of its three dimensional structure. This article describes mutation of the MAV proteinase which resulted not only in an alteration of its substrate specificity but also in an increase of its enzymic activity — a rare case in protein engineering.