interferENZY: A Web-Based Tool for Enzymatic Assay Validation and Standardized Kinetic Analysis.

Enzymatic assays are widely employed to characterize important allosteric and enzyme modulation effects. The high sensitivity of these assays can represent a serious problem if the occurrence of experimental errors surreptitiously affects the reliability of enzyme kinetics results. We have addressed this problem and found that hidden assay interferences can be unveiled by the graphical representation of progress curves in modified reaction coordinates.

STRENDA DB: enabling the validation and sharing of enzyme kinetics data.

Standards for reporting enzymology data (STRENDA) DB is a validation and storage system for enzyme function data that incorporates the STRENDA Guidelines. It provides authors who are preparing a manuscript with a user-friendly, web-based service that checks automatically enzymology data sets entered in the submission form that they are complete and valid before they are submitted as part of a publication to a journal.

Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.

Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin.

Probing the activity modification space of the cysteine peptidase cathepsin K with novel allosteric modifiers.

Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme.

A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods.

Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis.

Zymogen activation of neurotrypsin and neurotrypsin-dependent agrin cleavage on the cell surface are enhanced by glycosaminoglycans.

The serine peptidase neurotrypsin is stored in presynaptic nerve endings and secreted in an inactive zymogenic form by synaptic activity. After activation, which requires activity of postsynaptic NMDA (N-methyl-D-aspartate) receptors, neurotrypsin cleaves the heparan sulfate proteoglycan agrin at active synapses. The resulting C-terminal 22-kDa fragment of agrin induces dendritic filopodia, which are considered to be precursors of new synapses.

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates.

Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.

Human caspases in vitro: expression, purification and kinetic characterization.

A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer.

Clusterin is a specific stabilizer and liberator of extracellular cathepsin K.

The cysteine peptidase cathepsin K is a major player in extracellular proteolysis. Here we describe the identification of the multifunctional extracellular chaperone clusterin as a cathepsin K-binding protein. Clusterin increases the stability of cathepsin K in dilute solution and in the presence of high protein concentration.

Paradoxical interactions between modifiers and elastase-2.

The serine endopeptidase elastase-2 from human polymorphonuclear leukocytes is associated with physiological remodeling and pathological degradation of the extracellular matrix. Glycosaminoglycans bound to the matrix or released after proteolytic processing of the core proteins of proteoglycans are potential ligands of elastase-2. In vitro, this interaction results in enzyme inhibition at low concentrations of glycosaminoglycans.

Conformational flexibility and allosteric regulation of cathepsin K.

The human cysteine peptidase cathepsin K is a key enzyme in bone homoeostasis and other physiological functions. In the present study we investigate the mechanism of cathepsin K action at physiological plasma pH and its regulation by modifiers that bind outside of the active site. We show that at physiological plasma pH the enzyme fluctuates between multiple conformations that are differently susceptible to macromolecular inhibitors and can be manipulated by varying the ionic strength of the medium.

Calmodulin is a nonessential activator of secretory phospholipase A(2).

Ammodytoxins are presynaptically neurotoxic snake venom group IIA secreted phospholipase A(2) enzymes that interact specifically with calmodulin in the cytosol of nerve cells. We show that calmodulin behaves as an activator of ammodytoxin under both nonreducing and reducing (cytosol-like) conditions by stimulating its enzymatic activity up to 21-fold. Kinetic analysis, using a general modifier mechanism, and surface plasmon resonance measurements reveal that calmodulin influences both the catalytic and the vesicle binding properties of the enzyme without affecting its calcium binding properties.

Simultaneous interaction of enzymes with two modifiers: reappraisal of kinetic models and new paradigms.

Enzyme activity can be modulated by the concurrent action of two modifiers, either activators or inhibitors. The kinetic mechanisms for the interaction of the individual modifiers with the target enzyme can change considerably when two modifiers bind simultaneously. We illustrate a general equation for this kind of interactions, which can unambiguously describe the behavior of activators and inhibitors acting by any combination of classical kinetic mechanisms.

3-Fluoro-2,4-dioxa-3-phosphadecalins as inhibitors of acetylcholinesterase. A reappraisal of kinetic mechanisms and diagnostic methods.

A systematic survey of the acetylcholine-mimetic 2,4-dioxa-3-phosphadecalins as irreversible inhibitors of acetylcholinesterase revealed hitherto overlooked properties as far as the kinetic mechanisms of interaction are concerned. As a support to past and future work in this field, we describe the kinetics of eight reaction schemes that may be found in irreversible enzyme modification and compare them with two mechanism of reversible, slow-binding inhibition. The relevant kinetic equations and their associated graphical representations are given for all mechanisms, and concrete examples illustrate their practical use.

A double-headed cathepsin B inhibitor devoid of warhead.

Most synthetic inhibitors of peptidases have been targeted to the active site for inhibiting catalysis through reversible competition with the substrate or by covalent modification of catalytic groups. Cathepsin B is unique among the cysteine peptidase for the presence of a flexible segment, known as the occluding loop, which can block the primed subsites of the substrate binding cleft. With the occluding loop in the open conformation cathepsin B acts as an endopeptidase, and it acts as an exopeptidase when the loop is closed.

Purification and enzymological characterization of murine neurotrypsin.

An increasing number of studies indicate that serine proteases play an important role in structural plasticity associated with learning and memory formation. Neurotrypsin is a multidomain serine protease located at the presynaptic terminal of neurons. It is thought to be crucial for cognitive brain functions.

Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation.

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin-deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites.

Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism.

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition.

Interaction between human cathepsins K, L, and S and elastins: mechanism of elastinolysis and inhibition by macromolecular inhibitors.

Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung.

Regulation of human cathepsin B by alternative mRNA splicing: homeostasis, fatal errors and cell death.

One of the control mechanisms of cathepsin B biosynthesis and trafficking operates through alternative splicing of pre-mRNA. An mRNA lacking exon 2 is more efficiently translated than that containing all exons, and may be responsible for elevated biosynthesis and enzyme routing to the extracellular space, with critical consequences for connective tissue integrity in pathologies such as cancer and arthritis. mRNA missing exons 2 and 3 encodes a truncated procathepsin B form that is targeted to mitochondria.

Dual concentration-dependent activity of thyroglobulin type-1 domain of testican: specific inhibitor and substrate of cathepsin L.

The thyroglobulin type-1 (Tg-1) domain is a protein module that occurs in a variety of secreted and membrane proteins and is recognised as a potent inhibitor of cysteine peptidases. We present here some properties of the Tg-1 domain of human testican, a modularly organised proteoglycan secreted mainly by brain cells, the exact in vivo function of which is not yet clear. The domain was prepared as a recombinant protein in a Pichia pastoris expression system and its activity was demonstrated by specific and selective inhibition of cathepsin L (K(i) =0.

Folding competence of N-terminally truncated forms of human procathepsin B.

Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B.

Exon skipping of cathepsin B: mitochondrial targeting of a lysosomal peptidase provokes cell death.

The alternatively spliced messenger RNA of the human cysteine peptidase cathepsin B missing exons 2 and 3 encodes a truncated form of the enzyme lacking the signal peptide and part of the inhibitory propeptide. This deletion results in a new N-terminal leader sequence characteristic of proteins predestined for transport into mitochondria. We determined enzyme targeting to intracellular organelles by transfecting HeLa cells with constructs containing segments of variable length of the N terminus of truncated cathepsin B fused to green fluorescent protein.

Monomeric and dimeric bZIP transcription factor GCN4 bind at the same rate to their target DNA site.

Basic leucine zipper (bZIP) transcription factors are dimeric proteins that recognize dyadic and mostly palindromic DNA sites. Dimerization of bZIP transcription factor GCN4 is linked to the folding of its C-terminal leucine zipper domain. However, monomeric GCN4, lacking a folded leucine zipper, also recognizes the DNA site with dimerization taking place on the DNA.