Activation Mechanism of Pro-astacin: Role of the Pro-peptide, Tryptic and Autoproteolytic Cleavage and Importance of Precise Amino-terminal Processing

Yiallouros, Irene; Kappelhoff, Reinhild; Schilling, Oliver; Wegmann, Frank; Helms, Mike W.; Auge, Astrid; Brachtendorf, Gertrud; Berkhoff, Eva Große; Beermann, Bernd; Hinz, Hans‐Jürgen; König, Simone; Peter‐Katalinić, Jasna; Stöcker, Walter

doi:10.1016/s0022-2836(02)01102-6

Cited by 47 publications

(50 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The prosegment is elongated and stabilized through several intramolecular interactions. It runs across the front surface of the mature enzyme moiety blocking the cleft, which is consistent with previous functional studies (32) (Fig. 2, B and E).…”

Section: Msupporting

confidence: 92%

“…In contrast to other (metallo)peptidases, it does not have an intramolecular chaperone function in astacin, as revealed by refolding experiments with the heterologously expressed recombinant mature protein (32,48,49). The prosegment is elongated and stabilized through several intramolecular interactions.…”

Section: Mmentioning

confidence: 94%

“…Protein Preparation and Crystallization-Recombinant proastacin E93MA/I91ML (UniProt Q9U918; numbering is based on the mature enzyme (M); see below) was expressed in Escherichia coli BL21(DE3) cells as inclusion bodies, purified by Ni 2ϩ -nitrilotriacetic acid affinity chromatography, and folded by dilution and removal of reducing agents and guanidinium chloride as described (32). The resulting protein in 50 mM AMPSO buffer, pH 9.0, was concentrated in Amicon Ultra-15 centrifugal filter units of 10 kDa cut-off (Millipore, Bad Schwalbach, Germany) to a volume of about 1 ml.…”

Section: Methodsmentioning

confidence: 99%

“…A Novel Two-step Mechanism of Activation-Based on biochemical studies, proastacin activation in the crayfish is understood to be a twostep process entailing removal and degradation of the prosegment (32). In addition, maturation involves conformational changes in the first six residues of mature astacin, Ala 1M -Asp…”

Section: Mmentioning

confidence: 99%

“…The 15-residue signal peptide is removed during secretion, and the proenzyme, which comprises a 34-residue prosegment and a 202-residue catalytic domain, is only transiently found within the ducts between the hepatopancreas and the stomach. Once in the stomach, the zymogen is activated, and the mature enzyme participates in collagenolysis and gelatinolysis during digestion (32). Optimal astacin substrates comprise at least five amino acids with small aliphatic residues in PЈ 1 , proline in PЈ 2 , bulky hydrophobic residues in PЈ 3 , and basic residues in P 1 and P 2 (peptide substrate and enzyme subsite nomenclature according to Refs.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Proenzyme Structure and Activation of Astacin Metallopeptidase

Guevara

Yiallouros

Kappelhoff

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the activesite cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a "cysteine switch" mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its prosegment is the shortest structurally reported for a metallopeptidase, and it has a unique structure. It runs through the active-site cleft in reverse orientation to a genuine substrate. Moreover, a conserved aspartate, projected by a wide loop of the prosegment, coordinates the zinc ion instead of the catalytic solvent molecule found in the mature enzyme. Activation occurs through two-step limited proteolysis and entails major rearrangement of a flexible activation domain, which becomes rigid and creates the base of the substrate-binding cleft. Maturation also requires the newly formed N terminus to be precisely trimmed so that it can participate in a buried solvent-mediated hydrogen-bonding network, which includes an invariant active-site residue. We describe a novel mechanism for latency and activation, which shares some common features both with other metallopeptidases and with serine peptidases.The proteolytic activity of most metallopeptidases (MPs) 3 is regulated, and it is only exerted where and when required (1). Such control may occur through modulation of gene expression, compartmentalization, allostery, or inhibition by protein inhibitors. Another regulatory mechanism is zymogenic latency, which is provided by mostly N-terminal prosegments. These block access of substrates to the active-site cleft, and they are removed by limited proteolysis during maturation (2, 3). Such prosegments often fold independently and guide on their part the folding process of the cognate protease domains. They may also act as intramolecular chaperones or inhibitors of the mature enzymes in trans and in intracellular sorting of the zymogen (2). Therefore, the study of the molecular mechanisms by which MPs maintain latency is indispensable to an understanding of their basic mode of action. It also paves the way for the design of inhibitors that mimic the latent state so as to modulate MP activity as part of therapeutic approaches. Detailed three-dimensional structural information can contribute much to this understanding (4). However, among the MPs, only funnelins, lysostaphins, thermolysins, and matrix metalloproteinases (MMPs) have been structurally analyzed for their zymogens. Results reveal that the mature enzyme moieties are already in a competent conformation. Notwithstanding, each group displays a distinct mechanism of latency maintenance (5-11).The metzincins...

show abstract

Section: Msupporting

confidence: 92%

Section: Mmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Mmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Proenzyme Structure and Activation of Astacin Metallopeptidase

Guevara

Yiallouros

Kappelhoff

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

Astacin

St��cker¹,

Yiallouros²

2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

The mono‐zinc endopeptidase astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of metalloproteinases. Some astacins are digestive enzymes of invertebrates, but the majority serve functions during embryonic development, pattern formation, and epithelial differentiation throughout the animal kingdom. The catalytic protease domain of the astacins is topologically similar to the corresponding domains of the matrix metalloproteinases (MMPs), the adamalysins/reprolysins/ADAMs (a disintegrin and metalloprotease), the bacterial serralysins, and several other groups of zinc peptidases. They have in common a conserved zinc binding sequence, HEXXHXXGXXH, containing three histidines and a catalytically important glutamic acid residue, which acts as a general base during catalysis. Furthermore, there is a strictly conserved methionine‐containing turn structure beneath the active site, the Met‐turn, which gave rise to the designation metzincins for this superfamily of metalloproteins. In astacin, in addition to the three histidine residues of the zinc binding motif, the metal is ligated by the Glu93‐bound ‘activated’ water and by Tyr149, positioned in the Met‐turn, resulting in a trigonal bipyramidal coordination sphere. This overview treats the structural basis for the function of astacin‐like proteases, including proenzyme activation, cleavage specificity, substrate and inhibitor kinetics, catalysis, and metal binding. It also pinpoints shared commonalities and subtle differences between astacin and other metzincins.

show abstract

Handling Metalloproteinases

2016

Self Cite

View full text Add to dashboard Cite

Substrate cleavage by metalloproteinases involves nucleophilic attack on the scissile peptide bond by a water molecule that is polarized by a catalytic metal, usually a zinc ion, and a general base, usually the carboxyl group of a glutamic acid side chain. The zinc ion is most often complexed by imidazole nitrogens of histidine side chains. This arrangement suggests that the physiological pH optimum of most metalloproteinases is in the neutral range. In addition to their catalytic metal ion, many metalloproteinases contain additional transition metal or alkaline earth ions, which are structurally important or modulate the catalytic activity. As a consequence, these enzymes are generally sensitive to metal chelators. Moreover, the catalytic metal can be displaced by adventitious metal ions from buffers or biological fluids, which may fundamentally alter the catalytic function. Therefore, handling, purification, and assaying of metalloproteinases require specific precautions to warrant their stability.

show abstract

Activation Mechanism of Pro-astacin: Role of the Pro-peptide, Tryptic and Autoproteolytic Cleavage and Importance of Precise Amino-terminal Processing

Cited by 47 publications

References 33 publications

Proenzyme Structure and Activation of Astacin Metallopeptidase

Proenzyme Structure and Activation of Astacin Metallopeptidase

Astacin

Handling Metalloproteinases

Contact Info

Product

Resources

About