Maltose-binding sites

Maltose-binding site as an affnity tag for the purification and the immobilization... 

Most of the acceptors, such as maltose, isomaltose, and methyl a-D-glucopy-ranoside, were apparent competitive inhibitors for dextransucrase, as determined by Lineweaver-Burke or Hanes-Woolf plots103 however, when higher sucrose concentrations were used in a Michaelis-Menten plot, the inhibition was not reversed for methyl a-D-glucopyranoside as it should have been for a competitive inhibitor.103 It was concluded that the acceptors were being bound at a site that was separate and distinct from the sucrose binding-sites. 

Scanning linker mutagenesis can be used to identify sites amenable to functional insertion of another protein. TEM /3-lactamase was inserted into three sites of the E. coli maltose binding protein (MalE) (Betton et al., 1997), previously identified by random insertion of a BamH I linker to tolerate small insertions (Duplay et al., 1987). In all three hybrids, both activities were found to be essentially that of the individual wild-type enzymes. 

D-fructose, D-ribose, and maltose were without effect. Equilibrium-dialysis analysis147 revealed one binding site for lactose on the abrin molecule, with an apparent association constant of 8 X 103 M l. 

The simplest modification is to place a destabilizing amino acid at the N-terminal end according to the N-end rule (Varshavsky, 1992). This can be done by expressing the protein as a fusion protein, e.g. with maltose binding protein behind a cleavage site specific for a rare-cutting protease, such as factor Xq. In this case the N-terminal amino acid can be any amino acid except proline. 

As a variant of the studies discussed above, Lecroisey et al.1] undertook an NMR study of the 11 amino acid C3 epitope from poliovirus VP1 following its insertion at eight different sites into maltose-binding protein (MBP) (mass 41 kDa). The insertion of this epitope at the various sites did not cause significant structural changes to the protein so as to affect its ability to function normally and bind maltose. From H NMR studies of these mutants, it was found that the epitope had significant conformational flexibility and that the mobility was maintained regardless of where it was placed into the protein.71 Consistent with this mobility, the epitope adopted no ordered conformation in any of the mutants. 

Fig. 3. Substrate binding site on Taka-amylase A deduced from electron density difference maps with the enzyme-maltose complex and model building. The seven saccharide binding sites are numbered. Presumed catalytic amino acids Asp-206 and Asp-297 surround the sessile glycoside bond. Glu-230 is considered as a possible catalytic amino acid as well because of its proximity to the reaction center. Adapted from Matsuura et al. (262) with permission from J. Biochem (Tokyo).

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