Biomedical subjects

B Turk

Publications and source records attributed to B Turk.

At least 19 recordsLinked to original sources

PubMed1999

Autocatalytic processing of recombinant human procathepsin B is a bimolecular process.

Cathepsin B and other lysosomal cysteine proteinases are synthesized as inactive zymogens, which are converted to their mature forms by other proteases or by autocatalytic processing. Procathepsin B autoactivation was shown in vitro at pH 4.5 to be a bimolecular process with K(s) and k(cat) values of 2.1+/-0.9 microM and 0.12+/-0.02 s(-1)6.0. However, in the presence of 0.5 microg/ml of dextran sulfate, relatively rapid processing is observed even at pH 6.5 (t(1/2) approximately 90 min), suggesting that glycosaminoglycans are involved in in vivo processing of lysosomal cysteine proteases.

Catalysis↗

PubMed1999

A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(4-10) x 10(3) M-1 s-1] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s-1]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s-1 vs 560-710 s-1). Whereas the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as compared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.

Adenosine Triphosphate↗

PubMed1999

Acidic pH as a physiological regulator of human cathepsin L activity.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.

Amino Acid Sequence↗

PubMed1999

Mutational analysis of two stefin A epitopes.

Stefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants - Glu33Lys, Asp61Gly and Asn62Tyr and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating thatthe two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.

Amino Acid Sequence↗

PubMed1999

Interaction of cystatin C variants with papain and human cathepsins B, H and L.

Recombinant human cystatin C and two of its mutants were expressed in Escherichia coli. The recombinant inhibitor was found to be identical to authentic cystatin C as judged by isoelectric focusing (pI 9.2) and kinetics of inhibition of papain and human cathepsins B, H and L. N-terminal truncation of 8 residues resulted in a decrease of isoelectric point (pI 7.8), but the inhibitory properties were similar to those of recombinant cystatin C, suggesting that Leu9 is a critical residue for the inhibition. The mutation of Trp106 to Ser, however, resulted in a decreased affinity of the inhibitor for the enzymes tested, with the largest effect on cathepsin B inhibition (approximately 100-fold increase in Ki).

Cathepsin B↗

PubMed1998

Revised definition of substrate binding sites of papain-like cysteine proteases.

A review of kinetic and structural data has enabled us to reconsider the definition of substrate binding sites in papain-like cysteine proteases. Only three substrate binding sites, S2, S1 and S1', involve main as well as side chain contacts between substrate and enzyme residues. Interactions between the enzymes and the substrate P3 and P2' residues are based on side chains (an exception is cathepsin B which is a carboxydipeptidase), so their interaction surface spreads over a relatively wide area. The location and definition of substrate binding sites beyond S3 and S2' is even more questionable.

Amino Acid Sequence↗

PubMed1997

The oligosaccharide side chain on Asn-135 of alpha-antithrombin, absent in beta-antithrombin, decreases the heparin affinity of the inhibitor by affecting the heparin-induced conformational change.

The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.

Antithrombin III↗

PubMed1997

Equistatin, a new inhibitor of cysteine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain.

It is well known that the activities of the lysosomal cysteine proteinases are tightly regulated by their endogenous inhibitors, cystatins. Here we report a new inhibitor of cysteine proteinases isolated from sea anemone Actinia equina. The inhibitor, equistatin, is an acidic protein with pI 4.7 and molecular weight of 14,129. It binds tightly and rapidly to cathepsin L (ka = 5.7 x 10(7) M-1 s-1, Ki = 0.051 nM) and papain (ka = 1.2 x 10(7) M-1 s-1, Ki = 0.57 nM). The lower affinity for cathepsin B (Ki = 1.4 nM) was shown to be due mainly to a lower second order association rate constant (ka = 0.04 x 10(6) M-1 s-1). The inhibitor is composed of 128 amino acids forming two repeated domains with 48% identity. Neither of the domains shows any sequence homology to cystatins, but they do show a significant homology to thyroglobulin type-1 domains. A highly conserved consensus sequence motif of Cys-Trp-Cys-Val together with conserved Cys, Pro, and Gly residues is present in major histocompatibility complex class II-associated p41 invariant chain, nidogen, insulin-like growth factor proteins, saxiphilin domain a, pancreatic carcinoma marker proteins (GA733), and chum salmon egg cysteine proteinase inhibitor. In each of the domains of the equistatin, the three residues are similarly conserved, and the sequences Val-Trp-Cys-Val and Cys-Trp-Cys-Val are present in domains a and b, respectively. We suggest that equistatin belongs to a new superfamily of protein inhibitors of cysteine proteinases named thyroglobulin type-1 domain inhibitors. This superfamily currently includes equistatin, major histocompatibility complex class II- associated p41 invariant chain fragment, and chum salmon egg cysteine proteinase inhibitor.

Amino Acid Sequence↗

PubMed1996

Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp.

Crystal structures of N-carbamoylsarcosine amidohydrolase (CSHase; EC 3.5.1.59) have been analyzed by X-ray diffraction methods with two different inhibitors bound to the active site at 2.28 A and 2.37 A resolution. The catalytic center of the enzyme could be identified on the basis of these structures. The four substrate binding sites are situated at the intersubunit interfaces of the compact dimers AB and CD of the homotetrameric enzyme. Both inhibitors inactivate the enzyme irreversibly through covalent binding of their aldehyde groups to the thiol group of the active-site cysteine residue Cys177. Within the identified substrate binding sites a number of residues from different subunits are involved in hydrogen bonding of the inhibitors. Two residues (Ala172 and Thr173) that form an unusual cis-peptide bond at the binding site are important components in fixing the examined inhibitors by hydrogen bonds. An electrochemical enzyme assay for CSHase was used to test the effect of inhibitors and substrate analogs on the enzyme's activity, revealing the high substrate specificity of CSHase. The intrinsic tryptophan fluorescence of CSHase increases strongly upon substrate and inhibitor binding. As most of the tryptophyl residues are located at the active sites, they are thus considerably affected by ligand binding. Fluorescence-detected stopped-flow measurements have been used to study the kinetics of glyoxylate and substrate binding to CSHase. Substrate and inhibitor binding could clearly be distinguished in the stopped-flow experiments. Inhibitor binding reveals at least three different elementary processes, whereas substrate binding is much faster and contains phases with different signs in amplitude.

Amidohydrolases↗

PubMed1996

Interaction of human cathepsin C with chicken cystatin.

Cathepsin C was purified from human spleen by a rapid procedure, which included homogenization, ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and finally affinity chromatography on chicken cystatin-Sepharose. The interaction between cathepsin C and chicken cystatin was further characterized. It was found to be accompanied by a maximum decrease in fluorescence emission intensity at 336 nm. Fluorescence titration showed that human cathepsin C can bind four chicken cystatin molecules. The 4:1 binding stoichiometry was confirmed by titration monitored by the loss of enzyme activity. A non-competitive-competitive type of inhibition was determined from a double-reciprocal Lineweaver-Burk plot with a Ki value of 0.22 nM for the non-competitive inhibition.

Animals↗

PubMed1996

High-molecular-weight kininogen binds two molecules of cysteine proteinases with different rate constants.

Fluorescence titrations showed that high-molecular-weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped-flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (kass,1 = 23.0 x 10(6) M-1 s-1 and kass,2 = 3.4 x 10(6) M-1 s-1). As for low-molecular-weight kininogen, comparison of these kinetic constants with previous data for intact low- and high-molecular-weight kininogen and the separated domains indicated that the faster-binding site is also the tighter-binding site and is that of domain 3, whereas the slower-binding, lower-affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M(r) of high-molecular-weight kininogen was determined to be 83,500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.

Animals↗

PubMed1996

Characterization of cystatin C from bovine parotid glands: cysteine proteinase inhibition and antiviral properties.

Cystatin C, a low Mr cysteine proteinase inhibitor was isolated from bovine parotid glands by a procedure which includes alkaline treatment of the homogenate, affinity chromatography, gel filtration and ion exchange chromatography. The purified inhibitor has a pl of 8.0 and Mr of 14500. The identity with bovine cystatin C from colostrum was confirmed by N-terminal sequence of the inhibitor and amino acid composition. Cystatin C rapidly (kass = 5.5 x 10(7) M-1s-1) and tightly inhibits papain (Ki = 0.02 nM), whereas its interaction with bovine cathepsin B is substantially weaker (Ki = 4.4 nM). Bovine cystatin C also shows a weak antiviral effect on poliovirus infected human Hela cells.

Amino Acid Sequence↗

PubMed1996

Inhibition of bovine cathepsins L and S by stefins and cystatins.

Inhibition of bovine cathepsins L and S by bovine stefin B, human stefins A and B and cystatin C was studied under pseudo-first-order conditions by continuous fluorimetric assay. All inhibitors formed very tight complexes with the enzymes (Ki < or = 29 pM). The binding was reversible (kdiss = 0.52 - 16.7 x 10(-4) s-1) and very fast (kass = 2.8 - 6.2 x 10(7) M-1 S-1). Cystatin C was the strongest inhibitor of the enzymes, but the affinity was too tight to be measured accurately by this method. Consistently weaker inhibition of cathepsin S by all the stefins is apparent due mainly to the higher dissociation rate constants.

Animals↗

PubMed1995

Oligomeric structure and substrate induced inhibition of human cathepsin C.

Cathepsin C has been purified from human kidney by a modified procedure. Human cathepsin C was isolated as pure protein with a pI close to 6.0. The enzyme was shown to have a molecular mass of 200 kDa and to consist of four identical subunits, each composed of three different polypeptide chains, two of them disulfide-bound. Their NH2-terminal amino acid sequences were determined. Two chains showed pronounced similarity with the heavy and light chains of other papain-like cysteine proteinases, whereas the third one corresponded to the prosequence of the enzyme, thus showing that a substantial part of the proregion remains bound in the mature enzyme. The kinetics of substrate hydrolysis deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at higher substrate concentrations. These data are explained by a sequential cooperative interaction model, where an enzyme molecule can bind up to four substrate molecules but where only the binary enzyme-substrate complex is catalytically active. Substrate inhibition was observed over the whole range of pH activity. From the pH activity profile it can be concluded that at least three ionizable groups with pKa values 4.2, 6.8, and 7.7 are involved in substrate hydrolysis. Human cathepsin C thus appears to differ qualitatively from other cysteine proteinases of different origin.

Amino Acid Sequence↗

PubMed1995

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Animals↗

PubMed1995

Identification of bovine stefin A, a novel protein inhibitor of cysteine proteinases.

For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated M(r) of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (kass = 9.6 x 10(6) M-1.s-1, Ki = 29 pM). The binding to cathepsin H was also rapid (kass = 2.1 x 10(6) M-1.s-1), but weaker (Ki = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (kass = 1.4 x 10(5) M-1.s-1), but still tight (Ki = 1.9 nM).

Amino Acid Sequence↗

PubMed1995

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.

Amino Acid Sequence↗

PubMed1995

Regulation of the activity of lysosomal cysteine proteinases by pH-induced inactivation and/or endogenous protein inhibitors, cystatins.

The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).

Animals↗