Enzyme activity databases

As with chemical synthesis, the first step when prospecting for a particular biotransformation is to perform a literature search to check whether a suitable precedent has been described. Extensive technical literature resources in the public domain provide both examples of specific enzyme-catalysed reactions and descriptions of transformations where enzyme activity is inferred if not explicitly described. Currently, searches of online databases such as PubMed reveal over 2000 new publications per annum in the subject of enzyme catalysis (excluding reviews). 

The 4-coumarate CoA ligase (4CL EC 6.2.1.12) enzyme activates 4-coumaric acid, caffeic acid, ferrulic acid, and (in some cases) sinapic acid by the formation of CoA esters that serve as branch-point metabolites between the phenylpropanoid pathway and the synthesis of secondary metabolites [46, 47]. The reaction has an absolute requirement for Mg " and ATP as cofactors. Multiple isozymes are present in all plants where it has been studied, some of which have variable substrate specificities consistent with a potential role in controlling accumulation of secondary metabolite end-products. Examination of a navel orange EST database (CitEST) for flavonoid biosynthetic genes resulted in the identification of 10 tentative consensus sequences that potentially represent a multi-enzyme family [29]. Eurther biochemical characterization will be necessary to establish whether these genes have 4CL activity and, if so, whether preferential substrate usage is observed. 

Citrus species are well-known for their accumulation of flavone- and flavanone-glycosides, and thus should contain all of the enzyme activities necessary for the synthesis of these compounds. Two tentative consensus sequences for FNS-II have been identified by in silico analysis of the CitEST database, apparently representing the first identification of putative FNS-II genes in this genus [29]. Biochemical determination of function and analysis of the proteins encoded by these genes will be an important step toward elucidating flavone synthesis in Citrus. 

Fig. 34. Glu-72- Zn interactions in native carboxypeptidase A and in carboxypep-tidase A-inhibitor complexes (inhibitors have been reviewed by Christianson and Lipscomb, 1989). When substrates or inhibitors bind to the enzyme active site and interact with the zinc ion, the interaction of the metal with Glu-72 tends from bidentate toward uniden-tate coordination. The flexibility of protein-zinc coordination may be an important aspect of catalysis in this system, and the Glu-72->Zn - coordination stereochemistry observed here is consistent with the stereochemical analysis of carboxylate-zinc interactions from the Cambridge Structural Database (Carrell et al., 1988 see Fig. 4).

The ENZYME database at http //www.expasy.ch/enzyme/ provides information on EC number, name, catalytic activity, and hyperlinks to sequence data of enzymes. The 3D structures of enzymes can be accessed via Enzyme Structures Database at http //www.biochem.ucl.ac.uk/bsm/enzyme/index.html. Some other enzyme databases are listed in Table 7.1. 

The ENZYME nomenclature database (Figure 7.3) of ExPASy (Expert Protein Analysis System) at http //www.expasy.ch/enzyme/ can be searched by entering EC number or enzyme names. The query returns information on EC number, enzyme name, catalytic activity, cofactors (if any) and pointers to Swiss-Prot sequence, ProSite, and human disease(s) of the enzyme deficiency. 

Figure 7.8. The substrate interaction at the active site of an enzyme. The interaction of tetra-A/,A/,A/,A/-acetylchitotetraose (NAG4) with amino acid residues at the active site of lysozyme (ILZC.pdb) can be viewed/saved at PDBsum server (Enyme Structure Database->PDBsum->LIGPLOT of interactions under Ligand) linked to the Enzyme Structure Database.

Search the Enzyme Structure Database for y-chymotrypsin active site (by the aid of the active-site-modified enzyme or active-site-specific inhibitor-enzyme complex) to identify and depict (save pdb file) the catalytic triad of y-chymotrypsin. 

Prior to the start of any experimental substrate finding activity, databases should be mined. A tremendous amount of information about proteases, substrates, inhibitors, and structures can be retrieved from two searchable databases MEROPS (Rawlings et al., 2006) (http //merops.sanger. ac.uk) and BRENDA (www.brenda-enzymes.de), that serve as good starting points for assay development in many cases. These databases are available to the public and should be consulted as primary sources of information. 

The complex nature and interconnectivity of plant cell wall polymers preclude straightforward enzymatic digestion. There are dozens of enzyme families involved in plant cell wall hydrolysis, including cellulases, hemicellu-lases, pectinases, and lignin-modifying enzymes. The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) has classified cellulases and hemicellulases, like all enzymes, into different classes based on activity. Table 33.2 and Table 33.3, compiled from the IUBMB enzyme nomenclature database (http //www.chem.qmul.ac.uk/iubmb/ enzyme/), list the IUBMB enzyme classifications for cellulases and hemicellulases.153... 

CAZy (2009) Carbohydrate-active enzymes, online database, http //www.cazy.org. 7 Sep... 

Protein motifs can represent, among other things, the active sites of enzymes. They can also identify protein regions involved in determining protein structure and stability. The PROSITE, BLOCKS, and PRINTS databases (3-5) contain hundreds of protein motifs corresponding to enzyme active sites, binding sites, and protein family signatures. Motifs can also be used to identify features that confer particular chemical characteristics (such as thermal stability) on proteins (6). Protein sequence motifs can also be used to classify proteins into families (5). 

A much more ambitious database that builds on the IUBMB classification is BRENDA, maintained by the Institute of Biochemistry at the University of Cologne. In addition to the data provided by the ENZYME database, the BRENDA curators have extracted a large body of information from the enzyme literature and incorporated it into the database. The database format strives to be readable by both humans and machines. The categories of data stored in BRENDA comprise the EC-number, systematic and recommended names, synonyms, CAS-registry numbers, the reaction catalyzed, a list of known substrates and products, the natural substrates, specific activities, KM values, pH and temperature optima, cofactor and ion requirements, inhibitors, sources, localization, purification schemes, molecular weight, subunit structure, posttranslational modifications, enzyme stability, database links, and last but not least an extensive bibliography. Currently, BRENDA holds entries for approximately 3500 different enzymes. 

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