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

Abstract The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

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A catalytically active high-Mr form of human cathepsin B from sputum

Biochemical Journal ◽

10.1042/bj2540693 ◽

1988 ◽

Vol 254 (3) ◽

pp. 693-699 ◽

Cited By ~ 23

Author(s):

D J Buttle ◽

B C Bonner ◽

D Burnett ◽

A J Barrett

Keyword(s):

Lysosomal Enzyme ◽

Active Site ◽

Cathepsin B ◽

Human Liver ◽

Cysteine Proteinase ◽

Truncated Form ◽

Physiological Importance ◽

Catalytically Active ◽

Human Cathepsin ◽

Chicken Cystatin

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.

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Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2′ and P3′ variations in extended substrates

Biochemical Journal ◽

10.1042/bj3470123 ◽

2000 ◽

Vol 347 (1) ◽

pp. 123-129 ◽

Cited By ~ 26

Author(s):

Fernada C. Vieira PORTARO ◽

Ana Beatriz F. SANTOS ◽

Maria Helena S. CEZARI ◽

Maria Aparecida JULIANO ◽

Luiz JULIANO ◽

...

Keyword(s):

Amino Acids ◽

Significant Influence ◽

Active Site ◽

Cathepsin B ◽

Cathepsin L ◽

Cysteine Proteinases ◽

Aminobenzoic Acid ◽

Site Analysis ◽

Hydrophobic Amino Acids ◽

Human Cathepsin

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S4 and S3 subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S2ʹ and S3ʹ. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P4, P3, P2ʹ and P3ʹ were made. The S4 to S2ʹ subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S3ʹ, indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S4, S3, S2ʹ and S3ʹ of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P2ʹ respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P3ʹ. Basic residues at P3 and P4 were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P2ʹ (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His111 (H111A) and His110 (H110A) of cathepsin B led to an increase in kcat values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

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Human Cathepsin B Is a Metastable Enzyme Stabilized by Specific Ionic Interactions Associated with the Active Site

Biochemistry ◽

10.1021/bi00253a019 ◽

1994 ◽

Vol 33 (49) ◽

pp. 14800-14806 ◽

Cited By ~ 61

Author(s):

Boris Turk ◽

Iztok Dolenc ◽

Eva Zerovnik ◽

Dusan Turk ◽

Franc Gubensek ◽

...

Keyword(s):

Active Site ◽

Cathepsin B ◽

Ionic Interactions ◽

Human Cathepsin

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The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B

Biochemical Journal ◽

10.1042/bj2830449 ◽

1992 ◽

Vol 283 (2) ◽

pp. 449-453 ◽

Cited By ~ 24

Author(s):

B Walker ◽

B M Cullen ◽

G Kay ◽

I M Halliday ◽

A McGinty ◽

...

Keyword(s):

Active Site ◽

Cathepsin B ◽

Solid Phase ◽

Detection System ◽

Kinetic Characterization ◽

Cysteine Proteinases ◽

Selective Labelling ◽

Wide Range ◽

Human Cathepsin ◽

Biological Milieu

In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyldiazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1.min-1 and approximately 7.8 x 10(4) M-1.min-1, compare very favourably with those values determined for the urethane-protected analogue benzyloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J. Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidyldiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase, even in a complex biological milieu containing additional classes of proteinases.

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