Active Site Conservation Patterns

Fig. 11.2. Schematic representation of the primary structure of secreted AChE B of N. brasiliensis in comparison with that of Torpedo californica, for which the three-dimensional structure has been resolved. The residues in the catalytic triad (Ser-His-Glu) are depicted with an asterisk, and the position of cysteine residues and the predicted intramolecular disulphide bonding pattern common to cholinesterases is indicated. An insertion of 17 amino acids relative to the Torpedo sequence, which would predict a novel loop at the molecular surface, is marked with a black box. The 14 aromatic residues lining the active-site gorge of the Torpedo enzyme are illustrated. Identical residues in the nematode enzyme are indicated in plain text, conservative substitutions are boxed, and non-conservative substitutions are circled. The amino acid sequence of AChE C is 90% identical to AChE B, and differs only in the features illustrated in that Thr-70 is substituted by Ser.

The active site of LADH contains an Asp-His-Zn triad (see Figure 11). This pattern is quite common in zinc-enzymes. The aspartate affects the structure, electronic properties and energetics of the active site and thus the catalytic activity. Indeed, Asp49 is conserved in all mammalian ADHs . 

It has been postulated that there are two distinct groups of p-CAs, differing in their pattern of sequence conservation, active site design, and possibly in their mechanism. The three zinc-bound ligands and the Asp/Arg pair are conserved in every p-CA sequence known. Two sets of sequences are observed for active site residues, viz. in higher plants (Gln-151, Ser-161, Ser-163, Phe-179, Val-184, and Tyr-205) and in... 

In addition to conventional sequence motifs (Prosite, BLOCKS, PRINTS, etc.), the compilation of structural motifs indicative of specific functions from known structures has been proposed [268]. This should improve even the results obtained with multiple (one-dimensional sequence) patterns exploited in the BLOCKS and PRINTS databases. Recently, the use of models to define approximate structural motifs (sometimes called fuzzy functional forms, FFFs [269]) has been put forward to construct a library of such motifs enhancing the range of applicability of motif searches at the price of reduced sensitivity and specificity. Such approaches are supported by the fact that, often, active sites of proteins necessary for specific functions are much more conserved than the overall protein structure (e.g. bacterial and eukaryotic serine proteases), such that an inexact model could have a partly accurately conserved part responsible for function. As the structural genomics projects produce a more and more comprehensive picture of the structure space with representatives for all major protein folds and with the improved homology search methods linking the related sequences and structures to such representatives, comprehensive libraries of highly discriminative structural motifs are envisionable. 

Figure 5-2. Typical conservation patterns of three protein classes. Residues invariant or conserved in more than 80% ofthe sequences are printed on a black or grey background, respectively. A Mainly nonpolar conservation in the UBA domain, a small protein domain that interacts preferentially with ubiquitin1781. B Invariant polar active site residues in the phospholipase D family1291. C Nearly invariant metal-binding residues in the HtpX/Ste24 family of Zn-containing metalloproteases.

From what was said in the previous paragraphs, it appears that the specific conservation pattern of a protein family can be used to predict whether the proteins are enzymes, bind metal ions, or rather have a structural or regulatory role. If the proteins are known to be enzymes, the conservation pattern can be used to predict which residues are part of the active site, and possibly also which catalytic mechanism is being used. For example, it would be straightforward to submit a family of structurally uncharacterized proteases to that type of analysis in order to find out whether they are serine proteases, aspartate proteases, metalloproteases, or if they belong to a different class. Moreover, it is possible to compare the family s conservation pattern with those of other, better characterized enzyme families this approach will be discussed in more detail in Sect. 5.6. 

The pattern entries of the PROSITE database are based on a regular expression syntax, which emphasises only the most highly conserved residues in a protein family, corresponding approximately to what is termed a conservation pattern in Sect. 5.3. In contrast to the other databases mentioned below, PROSITE patterns do not attempt to describe a complete domain or even protein, but rather try to identify the functionally most important residue combinations, which in enzymes typically correspond to the active site. As an example of the PROSITE syntax, K-x(l,2)-[DEj would mean a lysine residue, followed by one or two arbitrary residues, followed by a residue that is either aspartate or glutamate. When a sequence is compared with a library of such patterns, any pattern is found to be either present or absent, no intermediate scores are assigned. Currently, the PRO SITE pattern libraries contains approximately 1400 entries. 

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