Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion.

BACKGROUND: Cysteine proteases of the papain superfamily are synthesized as inactive precursors with a 60-110 residue N-terminal prosegment. The propeptides are potent inhibitors of their parent proteases. Although the proregion binding mode has been elucidated for all other protease classes, that of the cysteine proteases remained elusive. RESULTS: We report the three-dimensional structure of rat procathepsin B, determined at 2.8 A resolution. The 62-residue proregion does not form a globular structure on its own, but folds along the surface of mature cathepsin B. The N-terminal part of the proregion packs against a surface loop, with Trp24p (p indicating the proregion) playing a pivotal role in these interactions. Inhibition occurs by blocking access to the active site: part of the proregion enters the substrate-binding cleft in a similar manner to a natural substrate, but in a reverse orientation. CONCLUSIONS: The structure of procathepsin B provides the first insight into the mode of interaction between a mature cysteine protease from the papain superfamily and its prosegment. Maturation results in only one loop of cathepsin B changing conformation significantly, replacing contacts lost by removal of the prosegment. Contrary to many other proproteases, no rearrangement of the N terminus occurs following activation. Binding of the prosegment involves interaction with regions of the enzyme remote from the substrate-binding cleft and suggests a novel strategy for inhibitor design. The region of the prosegment where the activating cleavage occurs makes little contact with the enzyme, leading to speculation on the activation mechanism.

Alignment/phylogeny of the papain superfamily of cysteine proteases.

An alignment/phylogeny of the papain superfamily of cysteine proteases was created using an initial structure-based alignment followed by successive iterations of sequence alignment and phylogenetic inference. The iterative approach resulted in significant improvements in the alignment/phylogeny. There were three groups of cysteine proteases that were distantly related and which could be aligned against each other only in the active site regions: the papain group, which included such stereotypical cysteine proteases as cathepsins B, C, H, L and S; and the bleomycin hydrolase and calpain groups. There was one bacterial sequence in each of the bleomycin hydrolase and calpain groups. The former probably arose by lateral gene transfer, the latter possibly by direct evolution from an ancestral protease predating the eukaryote/prokaryote divergence. The phylogeny of the papain group indicated that many families diverged almost simultaneously early during eukaryotic evolution. In mammals there are at least 12 distinct families of cysteine proteases, possibly many more, including at least two as yet uncharacterized enzymes.

NMR structural studies of human cystatin C dimers and monomers.

Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, "trapped" dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H -15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.

Engineering of proteases and protease inhibition.

Proteases are unquestionably the single most studied class of enzymes and yet many questions still remain about their mechanisms and roles. Protein engineering offers the opportunity to provide some of the answers. In this review, recent advances towards the understanding of stability, mechanism, specificity and regulation of proteases and their inhibitors are outlined. In addition, the application of this increased understanding is also discussed.

Detection of covalent enzyme-substrate complexes of nitrilase by ion-spray mass spectroscopy.

Nitrilase from Rhodococcus ATCC 39484 was found to consist of two species of Mr 40,258 +/- 2 and 40,388 +/- 2 Da. When the enzyme was incubated with nitrile substrates and the reaction quenched with acid, higher Mr species were observed. The mass differences were consistent with addition of a substrate molecule to each species. These results represent the first reported demonstration that this, or any other nitrilase forms a covalent intermediate with its substrates. The observation that the intermediate, suggested to be either a thioimidate or an acylenzyme, can be trapped by acidification indicates that the rate of breakdown of the intermediate is rate-limiting.

Delineating functionally important regions and residues in the cathepsin B propeptide for inhibitory activity.

Synthetic peptides derived from the proregion of rat cathepsin B were used to identify functionally important regions and residues for cathepsin B inhibition. Successive 5 amino acid deletions of a 56 amino acid propeptide from both the N- and C-termini has allowed the identification of two regions important for inhibitory activity: the NTTWQ (residues 21p-25p) and CGTVL (42p-46p) regions. Alanine scanning of residues within these two regions indicates that Trp-24p and Cys-42p contribute strongly to inhibition, their replacement by Ala resulting in 160- and 140-fold increases in Ki, respectively.

The specificity of the S1' subsite of cysteine proteases.

The specificity of the S1' subsite of the cysteine proteases cathepsin B, L, S and papain has been investigated using a series of intramolecularly quenched fluorogenic substrates (Dansyl-Phe-Arg-AA-Trp-Ala) where the P1' amino acid (AA) has been varied. Taken individually, each enzyme displays a relatively broad S1' subsite specificity and this subsite cannot be considered as a primary site of specificity. Notable differences do exist however between the various proteases. Cathepsin B prefers large hydrophobic residues in the P1' position of a substrate while cathepsin L has an opposite trend, favoring amino acids with small (Ala, Ser) or long but non-branched (Asn, Gln, Lys) side chains. Cathepsin S and papain display a somewhat broader S1' subsite specificity.

Nature of papain products resulting from inactivation by a peptidyl O-acyl hydroxamate.

Mass spectrometry has been used to provide insights into the mechanism of inhibition of cysteine proteases by a hydroxylamine derivative, CBZ-Phe-Gly-NH-O-CO-(2,4,6-Me3)Ph. An oxidized form of papain resulting from the incubation of the enzyme with the peptidyl hydroxamate in the absence of a reducing agent has been identified as a sulfinic acid. The presence of a covalent enzyme-inhibitor complex of molecular mass consistent with a sulfenamide adduct of papain could also be detected by this method. Implications on the mechanism of inactivation of cysteine proteases by peptidyl hydroxamates are discussed.

Synthesis of amidrazones using an engineered papain nitrile hydratase.

To demonstrate the usefulness of an engineered papain nitrile hydratase as a biocatalyst, a peptide amidrazone was prepared by incubation of the nitrile MeOCO-Phe-Alanitrile with the Gln19Glu papain mutant in the presence of salicylic hydrazide as a nucleophile. The amidrazone results from nucleophilic attack by salicylic hydrazide at the imino carbon of the thioimidate adduct formed between the enzyme and the peptide nitrile substrate. Compared to wild-type enzyme, the engineered nitrile hydratase causes a better than 4000-fold increase in the rate of amidrazone formation and yields a product of much higher purity. The advantages over other nitrile-hydrolyzing enzymes and current limitations of the papain nitrile hydratase are discussed.

The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B.

Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological processes. It has been known for a long time that like other lysosomal cysteine proteases, cathepsin B becomes inactivated and undergoes irreversible denaturation at neutral or alkaline pH. However, the mechanism of this denaturation process remains mostly unknown up to this day. In the present work, nuclear magnetic resonance spectroscopy was used to characterize the molecular origin of the neutral-pH inactivation and the refolding barrier of human cathepsin B. Two forms of human cathepsin B, the native form with Cys-29 at the active site and a mutant with Cys-29 replaced by Ala, were shown to have well-folded structures at the active and slightly acidic condition of pH 5. Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three-dimensional structure at neutral pH conditions. In addition, replacement of Cys-29 by Ala renders the process of the urea denaturation of human cathepsin B completely reversible, in contrast to the opposite behavior of the wild-type cathepsin B. These results are very surprising in that replacement of one single residue, the active-site Cys-29, can eliminate the neutral-pH denaturation and the refolding barrier. We speculate that this finding may have important implications in understanding the process of pH-triggered inactivation commonly observed for most lysosomal cysteine proteases.

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide.

Selected fragments of the 62-residue proregion (or residues 1p-62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p-51p and 26p-51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p-51p fragment, but remained in the 26p-43p and 28p-43p peptide fragments. Residues 23p-26p or TWQ25A in the N-terminal 1p-29p fragment did not show cathepsin B-induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p-51p peptide. Both the 1p-29p and 36p-51p fragments lack a common set of hydrophobic residues 30p-35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p-51p, 26p-51p, 28p-43p and 26p-43p, but the same residues induce the aggregation of peptides 16p-36p and 1p-36p. The peptide fragment 26p-43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p-43p or 26p-43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full-length cathepsin B proregion.

Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.

Epoxysuccinyl dipeptides as selective inhibitors of cathepsin B.

Epoxysuccinyl dipeptide analogs of E-64 (R-EpsLeuPro-R') (Figure 1) have been synthesized with the carboxylate group on the epoxide ring either free (R = OH) or converted to an ester or an amide (R = EtO or i-BuNH) and with the C-terminal amino acid proline either blocked (R' = OBzl) or free (R' = OH). These compounds were used to investigate the recently reported selectivity of this type of inhibitor for the lysosomal cysteine protease cathepsin B. It was shown that derivatization of the carboxylate on the epoxide ring confers selectivity for cathepsin B over papain only when it is combined to a dipeptidyl moiety with a free negatively charged C-terminal residue. It is proposed that this selectivity reflects interactions with histidine residues on a loop located in the primed subsites of cathepsin B which provides a positively charged anchor for the C-terminal carboxylate group of the inhibitor. The primed subsite loop of cathepsin B is not found in other cysteine proteases of the papain family and offers a unique template for designing selectivity in cysteine protease inhibitors.

Aziridine analogs of [[trans-(epoxysuccinyl)-L-leucyl]amino]-4-guanidinobutane (E-64) as inhibitors of cysteine proteases.

Aziridine derivatives of E-64 have been synthesized, and their characterization against the cysteine proteases cathepsin B, cathepsin L, and papain is reported. The inhibition was found to be strongly pH-dependent, with maximum activity observed at pH 4, indicating that the protonated aziridinium ion form of the inhibitor is the more reactive form. At low pH, the peptide aziridine HO-(L)Az-Leu-NH-iAm inactivated papain with a second-order rate constant, kinac/Ki, of 7.0 x 10(4) M-1 s-1, a value very close to that observed with E-64 or with the corresponding epoxysuccinyl analog HO-(L)Eps-Leu-NH-iAm. This demonstrates that with the correct peptide sequence, aziridine analogs of E-64 can be good irreversible inhibitors of cysteine proteases. Substitution of the epoxysuccinyl moiety by an aziridine does not affect the specificity of inhibition against the three proteases used in this study. The D-diastereomer is the preferred (by 10-fold) diastereomer for the inhibition of cysteine proteases. The reactivity of both diastereomers of iBuNH-Az-LeuPro-OH against cathepsin B was also found to be much lower than that of iBuNH-(L)Eps-LeuPro-OH, which is a potent selective inhibitor of cathepsin B. These differences are attributed mainly to the presence of the protonated aziridine ring, which can modify the binding mode of aziridine analogs at the active site of cysteine proteases.

Papain in organic solvents: determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition.

Synthesis of N-CBZ-(N-Carbobenzoxy)-1-amino-acid methyl esters from N-CBZ-amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent K(m) value of CBZ-glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S(2) subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.

Local pH-dependent conformational changes leading to proteolytic susceptibility of cystatin C.

Cystatin C, a cysteine protease inhibitor, was subject to hydrolysis at two sites when complexed with papain and in the presence of excess papain. A pH-dependent cleavage at His-86 increases Asp-87 was observed, as well as a pH-independent one at Gly-4 increases Lys-5. His-86 increases Asp-87 hydrolysis increased with decreasing pH and was characterized kinetically. It could be described by a single ionization with pKa = 3.4 +/- 0.2 and (kcat./Km)max. = 1.4 (+/- 0.4) x 10(4) M-1.s-1 at I = 0.3 M. C.d. spectroscopy, also at I = 0.3 M, demonstrated a conformational change with pKa = 3.2 +/- 0.2, indicating that the pH-dependence of hydrolysis was due to a conformational change in cystatin C. At I = 0.15 M, the pKa of the conformational change observed by c.d. shifted to 4.1 +/- 0.1. This indicates that at physiological ionic strength of 0.15 M, a significant proportion of cystatin C complexed with protease would be in a proteolytically labile conformation over the pH range 4.5 to 5, which is encountered in lysosomes. This may constitute a mechanism for clearing inappropriately localized cystatins. A pH-dependent conformational variability in this region of the inhibitor could explain the differences in the X-ray crystallographic and n.m.r. structures of the homologous chicken cystatin. The ionic-strength dependence of ionization indicates a hydrophobic stabilization of the ionizable group. The lack of pH-dependence of hydrolysis at Gly-4 increases Lys-5, with kcat./Km = 220 +/- 41 M-1.s-1 in the pH range 3.89 to 7.96 was unexpected in light of the normal, bell-shaped pH-dependence of papain-catalysed hydrolyses. This may reflect a different rate-limiting step of cystatin C hydrolysis.

Mechanistic origin of the sigmoidal rate behaviour of rat liver hexokinase D ('glucokinase').

Two recent proposals to account for the kinetic co-operativity of hexokinase D ('glucokinase') from rat liver are examined. A model in which the deviations from Michaelis-Menten kinetics result from a random order of binding of the substrates [Pettersson (1986) Biochem. J. 233, 347-350] accounts satisfactorily for the behaviour as a function of glucose concentrations, but it also predicts observable substrate inhibition by MgATP, which is in fact not observed. An alternative proposal in which the deviations arise from recycling of an enzyme-MgADP complex [Pettersson (1986) Eur. J. Biochem. 154, 167-170] also accounts satisfactorily for some of the data, but the required enzyme-MgADP complex could not be detected in isotope-exchange measurements. Thus the mnemonical mechanism proposed originally [Storer & Cornish-Bowden (1977) Biochem. J. 165, 61-69], which explains the deviations in terms of a relatively slow interconversion between two forms of free enzyme, remains the most parsimonious explanation of the behavior of hexokinase D.