**9. Results**

*vitro* assay. *In vivo,* the McIIPa activity was shown to be simultaneous to the decrease of TSN

Another substrate found for a metacaspase was the already mentioned GAPDH. This protein was detected as a digest product of an YCA1 metacaspase‐enriched extract from *S. cere‐*

*in vitro*, the *in vivo* evaluation of the GAPDH performed during a comparison between the wild‐type and a mutant yeast strain, disrupted for YCA1, both under PCD triggering condition, showed a reduction of the enzyme levels on the wild‐type (resistant to PCD). The GAPDH is also a caspase substrate, but the cleavage again happens on different sites of those targeted by the metacaspases. Yet, this is another evidence for the existence of conserved molecular members of a PCD pathway in metazoan and in metacaspase‐bearing

Recently, as a remarkable effort, a proteome‐wide‐level study of *A. thaliana* seedlings, focusing on the identification of physiologic substrates of metacaspase 9, has been performed employing a digestome analysis strategy. Important features of the target proteins were prospected, and it was possible to map the frequencies of the amino acids sitting at the neighbourhood of the Arg or Lys P1‐specific cleavage sites. Along with other interesting features, the enzyme has shown a strong tendency to prefer acid residues, as Asp and Glu at the P1' position. Among the identified substrates, was phosphoenolpyruvate carboxykinase 1 (PEPCK1), a gluconeogenesis enzyme. This protein was shown to be cleaved *in vivo* in such a manner that its activity was enhanced, and thus, the glucose *de novo* synthesis pathway may be stimulated

**8. Molecular modelling of the metacaspase 4 from** *Glycine max*

For the comprehension of the structural organization of a type II metacaspase, the delimitation of the p20 and p10 domains of the metacaspase 4 from *G. max* was performed by our group, as well as the analysis of its catalytic amino acids residues and the motifs conservation with other metacaspases and caspases, through protein alignment. Also, the tridimensional structure of the protein was predicted. Metacaspase and caspase sequences of organisms from different taxa (**Table 1**) were aligned using the software Clustal X [83] (http://www.clustal.org/). The sequences were obtained from the National Center of Biotechnology Information (http://www. ncbi.nlm.nih.gov/) data bank and given a treatment for removal of prodomains and loops, for adjustment to the alignment. In this process, the works of Vercammen et al. [8] and of Uren et al. [32] were used as a guide to the delimitation of the domains and catalytic residues.

The p20 and p10 domains of the *G. max* metacaspase were confronted to the Protein Data Bank (http://www.wwpdb.org/) for the search of templates for molecular prediction employing the software Swiss Model [84] (http://swissmodel.expasy.org/). The visualization, the analysis, the validation and the improving of the protein structures were performed with the assistance

of the software NOC [85] (http://noch.sourceforge.net).

treatment. As recombinant YCA1 was shown to cleave GAPDH

activity, what was deduced from TUNEL positive embryonic cells [81].

O2

*visiae* cells subjected to H<sup>2</sup>

36 Enzyme Inhibitors and Activators

organisms [56].

during PCD [82].

**(type II metacaspase)**

#### **9.1. Protein alignment**

The initial alignment of caspases and metacaspases sequences with the whole protein sequences (**Figure 3**) divided them into two groups: sequences with larger pro‐domains and shorter loops; and sequences with smaller pro‐domains and larger loops. Using the position of the catalytic dyad His‐Cys as guide and after the removal of pro‐domains and loops, the core of p20 and p10 domains became evident. The number of amino acid residues counted 235 residues, being 146 from p20 and 85 from p10 domain. It was also possible to underline the approximate segment borders for the domains of the *G. max* metacaspase, and it was seen that, besides the conserved catalytic dyad position, their adjacent residues (on primary sequence) shared the same chemical nature, encompassing all the protein sequences, despite the phylogenetic distance. The same was seen for the residues close to these regions, considering the p20 segment. In relation to p10, its catalytic residues presented conserved position, despite the chemical divergence (Asp for metacaspases and Arg for caspases). This segment also revealed conservation along its extension, such as the Ser residues close to the catalytic site, amino acids with shorter lateral groups and polar residues. Concerning the prediction

of secondary structure organization, it was possible to determine that about 33% of the sequence can form α‐helix/3‐10 helix and that about 23% can take part of β‐sheet conformation (**Figure 3**).

#### **9.2. Molecular structure prediction of** *G. max* **metacaspase 4**

The search for a protein that could be applied as a template using the individual subunits from *G. max* metacaspase, resulted in only one choice with a significant sequence similarity rate for the p20 subunit: the chain A of a protein complex from *Geobacter sulfureduccens* (PDB‐3BIJ). The identity and similarity rates between the template (from the 60th to the 148th residue) and p20 were of 35 and 55%, respectively. As the analysis of tertiary structure of the subunits and the total protein sequences were compatible, even with the high primary structure difference, 3BIJ was used as a template for the construction of a structural model for metacaspase 4. Only one α‐helix from 3BIJ was removed for adjustment to the target protein sequence.

Concerning the established structural model (**Figure 4**), it was possible to note that the amino acids residues from the catalytic dyad (His/Cys) of *G. max* metacaspase were closer arranged, spatially. The contact Asp residue also kept this position, suggesting that they are, in fact, intimately associated to the enzymatic catalysis. The model also presented a tridimensional structure close to that of caspases and related proteins, with a core of β‐conformations originated from both p20 and p10 being encircled by α‐helixes, also originated from both domains. It totalized two β‐conformations on its central position which are originated from p10, while p20 contributed with tree of these secondary structures. Other two β‐sheets occupied the model extremity. With relation to the α‐helixes and 3–10 helixes, there are eight of those that encircled the β‐conformation core. Three of those are originated from p20; other two are from p10. The remaining helixes are disposed on the model extremities.

**Figure 4.** Structural model generated for metacaspase 4 of*Glycine max*. (A) Lateral and (B) top view from catalytic site. The p20 and p10 domains are colored in green and red respectively. The lateral chains from the catalytic amino acids are in evidence.
