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.

Identification of internal autoproteolytic cleavage sites within the prosegments of recombinant procathepsin B and procathepsin S. Contribution of a plausible unimolecular autoproteolytic event for the processing of zymogens belonging to the papain family.

The steps involved in the maturation of proenzymes belonging to the papain family of cysteine proteases have been difficult to characterize. Intermolecular processing at or near the pro/mature junction, due either to the catalytic activity of active enzyme or to exogeneous proteases, has been well documented for this family of proenzymes. In addition, kinetic studies are suggestive of a slow unimolecular mechanism of autoactivation which is independent of proenzyme concentration. However, inspection of the recently determined x-ray crystal structures does not support this evidence. This is due primarily to the extensive distances between the catalytic thiolate-imidazolium ion pair and the putative site of proteolysis near the pro/mature junction required to form mature protein. Furthermore, the prosegments for this family of precursors have been shown to bind through the substrate binding clefts in a direction opposite to that expected for natural substrates. We report, using cystatin C- and N-terminal sequencing, the identification of autoproteolytic intermediates of processing in vitro for purified recombinant procathepsin B and procathepsin S. Inspection of the x-ray crystal structures reported to date indicates that these reactions occur within a segment of the proregion which binds through the substrate binding clefts of the enzymes, thus suggesting that these reactions are occurring as unimolecular processes.

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.

Interdependency of sequence and positional specificities for cysteine proteases of the papain family.

The specificity of cysteine proteases is characterized by the nature of the amino acid sequence recognized by the enzymes (sequence specificity) as well as by the position of the scissile peptide bond (positional specificity, i.e., endopeptidase, aminopeptidase, or carboxypeptidase). In this paper, the interdependency of sequence and positional specificities for selected members of this class of enzymes has been investigated using fluorogenic substrates where both the position of the cleavable peptide bond and the nature of the sequence of residues in P2-P1 are varied. The results show that cathepsins K and L and papain, typically considered to act strictly as endopeptidases, can also display dipeptidyl carboxypeptidase activity against the substrate Abz-FRF(4NO2)A and dipeptidyl aminopeptidase activity against FR-MCA. In some cases the activity is even equal to or greater than that observed with cathepsin B and DPP-I (dipeptidyl peptidase I), which have been characterized previously as exopeptidases. In contrast, the exopeptidase activities of cathepsins K and L and papain are extremely low when the P2-P1 residues are A-A, indicating that, as observed for the normal endopeptidase activity, the exopeptidase activities rely heavily on interactions in subsite S2 (and possibly S1). However, cathepsin B and DPP-I are able to hydrolyze substrates through the exopeptidase route even in absence of preferred interactions in subsites S2 and S1. This is attributed to the presence in cathepsin B and DPP-I of specific structural elements which serve as an anchor for the C- or N-terminus of a substrate, thereby allowing favorable enzyme-substrate interaction independently of the P2-P1 sequence. As a consequence, the nature of the residue at position P2 of a substrate, which is usually the main factor determining the specificity for cysteine proteases of the papain family, does not have the same contribution for the exopeptidase activities of cathepsin B and DPP-I.

The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide.

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent pH-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of human cathepsin B by its propeptide is defined by slow binding kinetics with a Ki of 3.7 nM and at pH 4.0 by classical kinetics with a Ki of 82 nM. This pH dependency is essentially eliminated either by the removal of a portion of the enzyme's occluding loop through deletion mutagenesis or by the mutation of either residue Asp22 or His110 to alanine; e.g., the mutant enzyme His110Ala is inhibited by its propeptide with Ki's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His110Ala mutant the inhibition also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzyme, and it has been shown that this bridge stabilizes the occluding loop in its closed position [Nägler, D. K., et al. (1997) Biochemistry 36, 12608-12615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propeptide. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing the occluding loop in its closed conformation, the loop more effectively competes with the propeptide than at higher pH where deprotonation of His110 and the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thus suggesting a possible influence of occluding loop stability on the rate of processing.

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.

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.

Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.

Affinity purification and elimination of methionine oxidation in recombinant human cystatin C.

Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.

Active site titration of the tyrosine phosphatases SHP-1 and PTP1B using aromatic disulfides. Reaction with the essential cysteine residue in the active site.

Aromatic disulfides were found to inactivate truncated forms of the SHP-1 and PTP1B phosphatases by reaction with the essential active site cysteine residue. For truncated SHP-1 at pH 5.0, the reaction proceeded through an initial burst phase followed by a slower secondary phase. Our experiments demonstrated that the burst phase corresponded to the reaction of the aromatic disulfide with the active site cysteine. The magnitude of the burst phase was found to measure the active enzyme concentration, and the rate of the burst reflected the reactivity of the active site cysteine. The data were consistent with a mechanism in which an intramolecular disulfide is formed between the active site cysteine and a proximal cysteine during the burst reaction. Aromatic disulfides were found to react with the active site cysteines of full-length SHP-1 and truncated PTP1B also. Using vanadate to mask the active site cysteine, the active enzyme concentration could be assayed by comparing product yields for the reaction with aromatic disulfides in the presence and absence of vanadate at pH 8.0. These findings demonstrate the utility of aromatic disulfides as active site titrants and reactivity probes for tyrosine phosphatases.

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.

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.

Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases.

Within the papain family of cysteine proteinases few other residues in addition to the catalytic triad, Cys25-His159-Asn175 (papain numbering) are completely conserved [Berti & Storer (1995) J. Mol. Biol. 246, 273-283]. One such residue is tryptophan 177 which participates in a Trp-His-type interaction with the catalytic His159. In all enzymes of this class for which a three-dimensional structure has been reported, an additional highly conserved tryptophan, Trp181, also interacts with Trp177 via an aromatic-aromatic interaction in which the planes of the indole rings are essentially perpendicular. Also, both indole rings participate as pseudo-hydrogen bond acceptors in interactions with the two side chain amide protons of Asn175. Clearly, the proximity of Trp177 and Trp181 to the catalytic triad residues His159 and Asn175 and their network of interactions points to potential contributions of these aromatic residues to catalysis. In this paper, using cathepsin S, a naturally occurring variant that has a phenylalanine residue at position 181, we report the kinetic characterization of mutants of residues 175, 177, and 181. The results are interpreted in terms of the side chain contributions to catalytic activity and thiolate-imidazolium ion-pair stability. For example, the side chain of Asn175 has a major influence on the ion-pair stability presumably through its hydrogen bond to His159. The magnitude of this effect is modulated by Trp177, which shields the His159-Asn175 hydrogen bond from solvent. The His159-Trp177 interaction also contributes significantly to ion-pair stability; however, Trp181 and its interactions with Asn175 and Trp177 do not influence ion-pair stability to a significant degree. The observation that certain mutations at positions 177 and 181 result in a reduction of kcat/Km but do not appear to influence ion-pair stability probably reflects the contributions of these residues to substrate binding.

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.

Engineering nitrile hydratase activity into a cysteine protease by a single mutation.

A peptide nitrile hydratase activity has been engineered into the cysteine protease papain by a single carefully selected mutation at the active site of the enzyme. The papain variant Gln19Glu hydrolyzes the substrate MeOCO-PheAla-CN to the corresponding amide with a kcat/KM value of 1.15 x 10(3) M-1 s-1. The reaction leads to an accumulation of the corresponding amide, which is then further hydrolyzed to the acid by the natural amidase activity of the enzyme. The pH-dependency of the nitrile hydratase activity of Gln19Glu supports the involvement of the acid form of the Glu19 residue in the reaction. The wild type enzyme displays very weak nitrile hydratase activity, and the introduction of a glutamic acid residue in the oxyanion hole of papain causes the kcat at pH 5 to increase by a factor of at least 4 x 10(5). Peptide nitriles react with cysteine proteases to form thioimidates, and the role of the glutamic acid residue introduced at position 19 in the Gln19Glu enzyme is to participate in the acid-catalyzed hydrolysis of the thiomidate to the amide by the provision of a proton to form the more reactive protonated thioimidate. This dramatically decreases the energy barrier for the hydrolysis of the thioimidate, as shown by the impressive increase in kcat. The success of the rational approach undertaken is a consequence of the level of understanding of the basic catalytic properties of cysteine proteases of the papain family.

Regulation of protein tyrosine phosphatase 1C: opposing effects of the two src homology 2 domains.

The regulatory roles of the two src homology 2 (SH2) domains of protein tyrosine phosphatase 1C were investigated by comparing recombinant full-length PTP1C with mutants in which either the N-terminal SH2 (N-SH2) domain (PTP1C delta NSH2), the C-terminal SH2 (C-SH2) domain (PTP1C delta CSH2) or both SH2 domains were deleted (PTP1C delta NSH2 delta CSH2). This revealed that the SH2 domains have opposing and independent effects on activity: strong inhibition by N-SH2 (42-fold) and weak activation by C-SH2 (2.1-fold). C-SH2 caused activation across a wide pH range while N-SH2 inhibited most at neutral and high pH through a shift of the basic limb of the pH profile of kcat/Km, apparently via perturbation of an active-site pKa value. A phosphotyrosyl peptide derived from the erythropoietin receptor caused an approximately 30-fold activation of PTP1C and PTP1C delta CSH2 but had no effect on PTP1C delta NSH2 or PTP1C delta NSH2 delta CSH2, indicating that binding of this peptide to N-SH2 abolished its inhibition. Since C-SH2 separates N-SH2 from the catalytic domain in full-length PTP1C and activation is observed for PTP1C delta CSH2, it appears that the inhibitory effect of N-SH2 is independent of the position in the sequence and that intermolecular interactions may also be possible.

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.

Peptide aldehydes and nitriles as transition state analog inhibitors of cysteine proteases.

Enzymes efficiently catalyze reactions by stabilizing inherently unstable transition states. For cysteine proteases, part of the stabilization is provided by a region of the enzyme termed the oxyanion hole. Site-directed mutagenesis has been used to investigate further the role of the oxyanion hole of papain in the binding of putative transition state analog inhibitors of cysteine proteases. The dissociation constants Ki(obs) for inhibition of wild-type and mutant enzymes (Gln19Ala, Gln19Glu, and Gln19His) by the aldehyde Ac-Phe-Gly-CHO and the nitrile MeOCO-Phe-Gly-CN have been determined in the pH range 3.5-9.0. For the peptide nitrile inhibitor, mutation of Gln19 was found to cause important increases in Ki(obs), and thioimidate adducts with the papain mutants Gln19Ala and Gln19Glu are less stable by 1.4-2.4 kcal/mol. However, for the peptide aldehyde inhibitor, the mutations resulted in a small but significant increase in stability of the tetrahedral hemithioacetal adduct (0.4-1.2 kcal/mol). In that respect, the hemithioacetal formed between papain and a peptide aldehyde cannot be considered a good model of the transition state for cysteine protease-catalyzed reactions. The influence of the mutations on the pH dependency of inhibition also indicates that with respect to oxyanion hole interaction, the inhibition of papain by peptide nitriles is a process closer to that of substrate hydrolysis than is the inhibition by the corresponding peptide aldehydes. The nature of the intermediates and transition states in hydrolysis reactions catalyzed by cysteine proteases, as well as the use of enzyme-inhibitor adducts as their models, is discussed.

Structural and functional roles of asparagine 175 in the cysteine protease papain.

The role of the asparagine residue in the Cys-His-Asn "catalytic triad" of cysteine proteases has been investigated by replacing Asn175 in papain by alanine and glutamine using site-directed mutagenesis. The mutants were expressed in yeast and kinetic parameters determined against the substrate carbobenzoxy-L-phenylalanyl-(7-amino-4-methylcoumarinyl)- L-arginine. At the optimal pH of 6.5, the specificity constant (k(cat)/KM)obs was reduced by factors of 3.4 and 150 for the Asn175-->Gln and Asn175-->Ala mutants, respectively. Most of this effect was the result of a decrease in k(cat), as neither mutation significantly affected KM. Substrate hydrolysis by these mutants is still much faster than the non-catalytic rate, and therefore Asn175 cannot be considered as an essential catalytic residue in the cysteine protease papain. Detailed analyses of the pH activity profiles for both mutants allow the evaluation of the role of the Asn175 side chain on the stability of the active site ion pair and on the intrinsic activity of the enzyme. Alteration of the side chain at position 175 was also found to increase aggregation and proteolytic susceptibility of the proenzyme and to affect the thermal stability of the mature enzyme, reflecting a contribution of the asparagine residue to the structural integrity of papain. The strict conservation of Asn175 in cysteine proteases might therefore result from a combination of functional and structural constraints.

Processing of the papain precursor. The ionization state of a conserved amino acid motif within the Pro region participates in the regulation of intramolecular processing.

The cysteine protease papain is synthesized as a 40-kDa inactive precursor with a 107-amino-acid N-terminal pro region. Although sequence conservation in the pro region is lower than in the mature proteases, a conserved motif (Gly-Xaa-Asn-Xaa-Phe-Xaa-Asp-36, papain precursor numbering) was found within the pro region of cysteine proteases of the papain superfamily. To determinate the function to this conserved motif, we have mutagenized at random each of the 4 residues individually within the pro region of the papain precursor. Precursor mutants were expressed in yeast, screened according to their ability to be processed through either a cis or trans reaction, into mature active papain. Three classes of mutants were found. Non-functional propapain mutants of the first class are completely degraded by subtilisin indicating that they are not folded into a native state. Mutants of the second class were neutral with respect to cis and trans processing. The third class included mutants that mostly accumulated as mature papain in the yeast vacuole. They had mutations that had lost the negatively charged Asp-36 residues and a mutation that probably introduces a positive charge, Phe-38His. The precursor of the Phe-38His mutant could be recovered by expression in a vph1 mutant yeast strain which has a vacuolar pH of about 7. The Phe-38His propapain mutant has an optimum pH of autoactivation about one pH unit higher than the wild type molecule. These results indicate that the electrostatic status of the conserved motif participates in the control of intramolecular processing of the papain precursor.