Identification and characterization of functional intermediates of stem bromelain during urea and guanidine hydrochloride unfolding

By comparing changes in enzyme activity with changes in spectral features for stem bromelain (EC.3.4.22.32) in the absence and presence of urea, Guanidine hydrochloride and ethanol; four intermediate states could be identified: two activity-enhanced state obtained in the presence of 5 M urea and 2 M GnHCl, termed X and X', respectively, and a third, similarly active state closely resembling the native protein in the presence of 8-9 M urea, termed Y. The enhanced activity of these states is due to local conformational changes accompanied by increased dynamics in the active site. Further, the enzyme does not lose its activity after substantial tertiary structure changes in 8-9 M urea (Y state), suggesting that active site containing domain is more resistant to chemical denaturation than the other structural domain. This makes stem bromelain and in general cysteine proteases an exception to the hypothesis that active site is the most labile part of enzyme.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway.     

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.     

Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base

3-oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testosteroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through an intermediate dienolate. Residue Asp-38 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6. Both Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) >/= 9.5) function as hydrogen-bond donors to O-3 of the steroid, helping to stabilize the transition states. Mutation of the active-site base Asp-38 to the weakly basic Asn (D38N) has previously been shown to result in a >10(8)-fold decrease of catalytic activity. In this work, we describe the preparation and kinetic analysis of the Ala-38 (D38A) mutant. Unexpectedly, D38A has a catalytic turnover number (k(cat)) that is ca. 10(6)-fold greater than the value for D38N and only about 140-fold less than that for wild type. Kinetic studies as a function of pH show that D38A-catalyzed isomerization involves two groups, with pK(a) values of 4.2 and 10.4, respectively, in the free enzyme, which are assigned to Asp-99 and either Tyr-14 or Tyr-55. A mechanism for D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transition states provided by hydrogen bonding from both Tyr-14 and Tyr-55. This mechanism is supported by the lack of detectable activity of the D38A/D99N, D38A/Y14F, and D38A/Y55F double mutants.     

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I₁ (existing in 1.8–1.4 M urea) and I₂ (existing in 0.8–0.4 M urea). I₁ was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I₂ was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates (I_B). Comparison of the properties of these intermediates suggested that I_B in the kinetic process corresponded to I₁ in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.     

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.     

Kinetics of the reduction of horse heart ferricytochrome c. Ascorbate reduction in the presence and absence of urea

The reduction of horse heart cytochrome c with ascorbate in the absence of urea and in its presence, 0 to 8 M, pH 7.0, has been investigated using a stopped flow technique and the absorptivity at 550 nm as the monitoring probes, and by using the rate of oxidizability with molecular oxygen. Reduction is found to be consistent with a mechanism involving (i) a urea-dependent equilibrium step between an ascorbate-reducible and an irreducible form, with a [urea]1/2 of 7.5 M and a reversion rate constant of 0.05 +/- 0.02 s-1, (ii) the binding of ascorbate to cytochrome c, with a binding constant of 5.9 M-1 in the absence of urea which decreases to a value of 2.7 M-1 above 5.5 M urea, and (iii) a reduction step, with a urea-independent rate constant of 2.9 +/- 0.3 s-1. This scheme is interpreted in terms of an electron-transfer pathway involving neither the classical "adjacent" attack nor attack at the exposed heme edge, i.e. "remote" attack, but rather, through an alternate pathway involving binding at some site other than the heme crevice opening and a migration path of rather low electron-transfer efficiency. The urea-linked ascorbate reduction step is th X2 in equilibrium D step of the urea denaturation mechanism (Myer, Y. P., MacDonald L. H., Verma, B. C., and Pande, A. J. (1980) Biochemistry 19, 199-207), and the 9 M urea form, D, is the irreducible form. Form X2 and the other intermediate form, X1, are found to be reducible directly by ascorbate, and not through reversion to the native form of the protein. both the integrity of the heme crevice and the polypeptide-organized structures are of little importance as far as ascorbate reducibility is concerned, but the integrity of the structural and protein functional changes reflecting the X2 in equilibrium D step of the mechanism directly or indirectly determines the reducibility of the protein.     

Effect of metal binding on the structural stability of pigeon liver malic enzyme.

The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degrees C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn(2+), the urea denaturation pattern of malic enzyme changed to monophasic. Mn(2+) helped the enzyme to resist phase I urea denaturation. The [urea](0.5) for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn(2+) retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn(2+). These results indicate that the Mn(2+) has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.     

Crystal structure of cathepsin X: a flip-flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease

BACKGROUND: Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer's disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored. RESULTS: The crystal structure of human cathepsin X has been determined at 2.67 A resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy-monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. CONCLUSIONS: The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases.     

Competitive RT-PCR for studying gene expression in micro biopsies

Characterization of cathepsin B from buffalo kidney and goat spleen showed the presence of isozymes in case of the goat spleen (GSCB-I and GSCB-II) whereas cathepsin B from buffalo kidney exhibited only one form (BKCB). The molecular weights determined by SDS-PAGE for GSCB-I, GSCB-II, and BKCB were 25.7, 26.6 and 25.5 kDa respectively. The kinetic parameters (K_m and V_max) of GSCB-I showed close similarities with BKCB against -N-benzoyl-DL-arginine-2-napthylamide whereas GSCB-II was closer to the buffalo enzyme with regards to its activity against Z-Arg-Arg-MCA and Z-Phe-Arg-MCA. All the three enzymes had similar sensitivities towards urea, antipain and leupeptin. However, clear differences were observed in the inhibition patterns of the enzyme with iodoacetic acid and iodoacetamide. Differences in the kinetic, immunogenic and some catalytic properties of GSCB-I and II, which had similarities with regard to most of their physico-chemical properties, were considered to be due to the existenceof two isozyme forms in goat spleen cathepsin B preparations. Absence of such a multiplicity in forms of the enzyme from buffalo kidney was accordingly attributed to the absence of cathepsin B isozymes in this species. These observations taken together therefore, indicate a probable species/tissue dependence of cathepsin B.     

The Effect of Urea on Acid Phosphatase Deactivation

Acid phosphatase thermal deactivation follows a complex path consisting of an initial decay of the native enzyme towards an equilibrium distribution of two intermediate structures, mutually at equilibrium. This initial transition is followed by a final decay towards a completely inactive enzyme configuration.

All the relevant parameters (one equilibrium and two kinetic constants) of the phenomenon are environment-sensitive. It is shown that urea affects the deactivation, by increasing the rate of both structural transitions as well as the thermodynamics of the equilibrium between intermediate forms. For every urea concentration up to 2.4M, an equivalent temperature can be calculated that yields exactly the same activity versus time profile. The result suggests that enzyme deactivation is controlled by a single parameter. Entirely different environments, so long as they result in the same value of the latter, are therefore bound to produce the same deactivation profile.

Marked deviations from thermal equivalence become apparent at higher urea concentrations. Therefore, extremely high urea concentrations seems to give rise to a change in the deactivation mechanism.     

Analysis of protein folding by fast protein liquid chromatography. Modular domain folding of gamma-II-crystallin from calf eye-lens

Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.     

Characterization of recombinant human cathepsin B expressed at high levels in baculovirus.

The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper-glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kcat. In addition, the biophysical properties of the baculovirus-expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.     

Human cathepsin B1. Purification and some properties of the enzyme

1. Cathepsin B1 was purified from human liver by a method involving autolysis, fractional precipitation with acetone, adsorption on, and stepwise elution from, CM-cellulose and an organomercurial adsorbent, gel chromatography and finally equilibrium chromatography on CM-cellulose. 2. The early stages of the procedure, including the use of the organomercurial adsorbent, were suitable for the simultaneous isolation of cathepsin D. The two cathepsins were sharply separated on the organomercurial column, and particular attention was given to the method for the preparation and use of this adsorbent. 3. A method is described for the staining of analytical isoelectric-focusing gels for cathepsin B1 activity, as well as protein. By this method it was shown that cathepsin B1 was represented by at least six isoenzymes during the greater part of the purification procedure. After the gel-chromatography step this group of isoenzymes was obtained essentially free of other proteins, in good yield. The isoenzymes were resolved from this mixture by chromatography on CM-cellulose. The purified enzyme was stable for several weeks at slightly acid pH values in the absence of thiol compounds; it was unstable above pH7. 4. The pI values of the isoenzymes of cathepsin B1 extended from pH4.5 to 5.5, that of the major isoenzyme tending to increase from 5.0 to 5.2 during the purification procedure. Gel chromatography indicated a molecular weight of 27500 for all of the isoenzymes, whereas polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate gave a value of 24000. 5. An antiserum raised in sheep against the purified enzyme reacted specifically with the alkali-denatured molecule. Purified cathepsin B1 contained no material precipitable by an anti-(human cathepsin D) serum. 6. The enzyme hydrolysed several N-substituted derivatives of l-arginine 2-naphthylamide, as well as haemoglobin, azo-haemoglobin, azo-globin and azo-casein. Greatest activity was obtained near pH6.0. 7. The sensitivity of human cathepsin B1 to chemical inhibitors was generally similar to that of other thiol proteinases. The enzyme was inactivated by the chloromethyl ketones derived from tosylphenylalanine, tosyl-lysine, acetyltetra-alanine and acetyldialanylprolylalanine. 8. The hydrolysis of alpha-N-benzoyl-dl-arginine 2-naphthylamide by extracts of human liver at pH6 was attributable entirely to cathepsin B1.     

Structure-activity relationship in buffalo spleen cathepsin B.

Effect of pH, urea, and guanidine hydrochloride on the activity and structure of buffalo spleen cathepsin B was investigated. At alkaline pH, there was an irreversible loss of the structure as well as the activity of the buffalo enzyme. At acidic pH, however, the inactivation of the enzyme was reversible. The enzyme reversibly lost most of its activity at denaturant concentrations which did not cause a significant change in its secondary structure. The inactivation could be attributed to minor perturbations in the environment of the amino acid residue(s) at and/or around the active site of the enzyme. High urea/guanidine hydrochloride concentrations leading to the structural changes in cathepsin B made the inactivation process irreversible.     

Tests of the simple model of Lin and Brandts for the folding kinetics of ribonuclease A

L.-N. Lin and J.F. Brandts recently proposed a simple model for the folding kinetics of ribonuclease A in which folding intermediates are not detectable. We have tested the basic assumption of the simple model for the major unfolded species, which is produced by a slow isomerization (the "X in equilibrium Y reaction" according to Lin and Brandts) after unfolding. The simple model assumes that in refolding the slow Y----X reaction must occur before any folding can take place. We have measured the Y----X reaction during folding. Tyrosine-detected folding occurs before the Y----X reaction; the difference in rate between the Y----X reaction and folding monitored by tyrosine absorbance becomes large when the stabilizing salt 0.56 M (NH4)2SO4 is added. The simple model predicts that the kinetic properties of the X in equilibrium Y reaction in unfolded ribonuclease are the same as those of tyrosine-detected folding. We find, however, that the kinetics of the X in equilibrium Y reaction in unfolded ribonuclease are independent of urea concentration, whereas the rate of tyrosine-detected folding decreases almost 100-fold between 0.3 and 5 M urea, as reported by Lin and Brandts. We point out that the kinetic properties of the X in equilibrium Y reaction in unfolded ribonuclease are characteristic of proline isomerization.     

Kinetic evidence of microscopic states in protein folding.

Staphylococcal nuclease unfolds at acidic pHs and refolds at neutral pH. Previous kinetic analysis based on both the direct pH jump and the sequential pH jump, from a native condition (pH 7.0) to pHs beyond unfolding transition zones (pH 3.0 and pH 12), and vice versa, supports the mechanism, D3<-->D2<-->D1<-->N0, in which N0 is the native state and D's are the three substates of the denatured form [Chen, H.M., You, J.L., Markin, V.S., & Tsong, T.Y. (1990) J. Mol. Biol. 220, 771-778; Chen, H.M., Markin, V.S., & Tsong, T.Y. (1992) Biochemistry 31, 1483-1491]. Here we show that both the single- and the double-pH jump kinetics of folding and unfolding to the intermediate pHs (3.4-5.0, i.e., in the transition zone), in which both the native and the denatured states coexist, are not compatible with this simple sequential model. At 25 degrees C, log tau 1(-1) (for the D1<-->N0 step) and log tau 2(-1) (for the D2<-->D1 step) vs pH show a square root of-shaped dependence on the final pH, with minimal values (tau 1(-1) of 0.56 s-1 and tau 2(-1) of around pH 3.9. The third relaxation tau 3 (for the D3<-->D2 step, 35 s) was independent of pH in the range 3.4-8.5. The square root of-shaped dependence on pH of log tau 1(-1) and log tau 2(-1) cannot be reproduced by the above but can be accounted for if each of N0, D1, and D2 is composed of many microscopic states in rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase.

Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of approximately 33 kDa and pI 5.1-5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7-15.0 nM), but poorly or not at all by stefin B (Ki > 250 nM) and L-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1' position, although the enzyme cleaved all P1' residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat/Km approximately 5.0 x 103 M-1.s-1) were degraded approximately 25-fold less efficiently than the carboxypeptidase substrates (kcat/Km approximately 120.0 x 103 M-1.s-1).