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Surface Binding Site

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Surface Binding Sites

Figure 1. The barley α-amylase 1 in complex with maltoheptaose PDB ID 1rp8 [1]. Several of the key SBS residues are shown highlighted in yellow, while the maltoheptaose molecules are shown in orange. Note the relatively large distance from the active site, which is a common aspect of these sites.

A surface (or secondary) binding site (SBS) is a ligand binding site observed on the catalytic module of an enzyme, but outside of the active site itself (see Figure 1). For recent reviews on this topic, please see [2, 3, 4].

Detection and Occurrence

SBSs have been observed in the crystal structures of approximately 50 carbohydrate active enzymes, with about half of these enzymes belonging to the family GH13 (Table 1). Typically the enzymes found to possess one or more SBSs are active on polysaccharides, suggesting that SBSs are adaptations for dealing with longer substrates. X-ray crystallography has been the main method of detecting SBSs; however, NMR spectroscopy [5] and chemical labeling [6] have also been used in the detection of these sites. Examination of the SBS containing enzymes show that they frequently co-occur with carbohydrate-binding modules (CBMs), suggesting that these two methods of binding to a substrate are complementary rather than redundant [2]. In one example, the α-amylase SusG from Bacteroides thetaiotaomicron, both a CBM and an SBS were found to contribute to binding to starch granules [7].

Roles of SBSs in Enzyme Function

Detailed analyses of SBSs have only been carried out in a few cases; however, in each of these cases they have been found to be important for the function of the enzyme. Various proven and speculated roles have been recently reviewed [2, 3, 4]. In general the proposed roles of SBSs include: i) serving as an extension of the active site, guiding a substrate strand to the active site or maintaining binding to a polysaccharide strand to allow processivity, ii) acting as an allosteric regulator, with binding at the SBS affecting the properties of the active site, iii) serving as a pseudo-CBM, by targeting the enzyme to the substrate, anchoring the enzyme to the cell wall or disrupting the substrate (see the carbohydrate-binding modules page for more details on their functional roles). As an illustrative example, the two SBSs of the barley α-amylase 1 (named SBS1 and SBS2) [1] seem to fall into categories i) and iii). SBS1 is particularly important for the binding of the enzyme to starch granules [8], while SBS2 is more important for the activity of the enzyme on amylopectin, lowering the apparent KM for this substrate [9]. A good example of ii) is seen in the amylomaltase from Thermus aquaticus, where binding to the SBS changes the active site, thereby altering the substrate profile of the enzyme [10].

Studying SBSs

The study of SBSs is often complicated by the presence of multiple SBSs in a given catalytic module, substrate binding in the active site, or the presence of a CBM. Various techniques have been used to dissect contributions to SBSs such as the use of mutations, and substrates that do not bind at the active site [8] or the use of covalent inhibitors to block the active site [5, 11]. A variety of techniques have proven useful for studying SBSs, including surface plasmon resonance, isothermal titration calorimetry, affinity electrophoresis and adsorption assays (the use of these techniques and others is summarized in [2]).

Table 1: Glycoside hydrolase enzyme families for which an enzyme with an SBS has been identified.
Family # of Enzymes as of 2015-02-17 Example Structure Reference(s)
GH1 2 1uyq Unpublished
GH5 2 2pc8 [12]
GH8 1 2b4f [13]
GH10 2 1goq [14, 15]
GH11 3 2qz3 [5, 16]
GH13 24 1rp8 [1, 2, 3]
GH14 1 1b9z [17]
GH15 1 2f6d [18]
GH16 1 1urx [19]
GH19 1 3cql [20]
GH27 1 3hg2 [21]
GH31 1 3nqq Unpublished
GH34 1 1mwe [22]
GH55 1 4pf0 [23]
GH57 1 3n98 [24]
GH63 1 3c67 [25]
GH77 1 1esw [26]
GH120 1 3vsv [27]


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  18. Sevcík J, Hostinová E, Solovicová A, Gasperík J, Dauter Z, and Wilson KS. Structure of the complex of a yeast glucoamylase with acarbose reveals the presence of a raw starch binding site on the catalytic domain. FEBS J. 2006 May;273(10):2161-71. DOI:10.1111/j.1742-4658.2006.05230.x | PubMed ID:16649993 | HubMed [Sevcik2006]
  19. Allouch J, Helbert W, Henrissat B, and Czjzek M. Parallel substrate binding sites in a beta-agarase suggest a novel mode of action on double-helical agarose. Structure. 2004 Apr;12(4):623-32. DOI:10.1016/j.str.2004.02.020 | PubMed ID:15062085 | HubMed [Allouch2004]
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