Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae

Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae

“…Yamamoto and coworkers reported that maltose act as a competitive inhibitor of Ima1p [13], which agreed with its ability to bind in the substrate-binding site [13,14]. This study was nevertheless carried out at concentrations of isomaltose (50-500 mM) that, according to our results, were inhibitory for Ima1p.…”

“…This work completed the biochemical and enzymological analysis that was carried out on the isomaltase isoenzyme 1 encoded by S. cerevisiae IMA1 [5,13,14] and shed light on several discrepancies in published data on substrate utilization by this type of enzyme in yeast [8][9][10][11][12]. We confirmed the preference of the four Ima proteins for the a-1,6 disaccharides isomaltose and palatinose [6,15], and despite some activity on isomaltotriose, they were totally inactive on longer isomalto-oligosaccharides (IMOs).…”

“…A gene encoding an isomaltase was first isolated from a Saccharomyces cerevisiae cDNA library and turned out to be YGR287c encoding a 589 amino acid protein able to hydrolyze isomaltose and a-MG but totally inactive on maltose [5]. The crystal structure of this protein was solved, underlying the importance of some amino acids in the structure of the active site and explaining its specificity for isomaltose [13,14]. More recently, we found with another group that the yeast S288c genome actually bears five related coding sequences located in the subtelomeres of different chromosomes that compose the IMA multigene family [6,15].…”

Reconstruction of Ancestral Metabolic Enzymes Reveals Molecular Mechanisms Underlying Evolutionary Innovation through Gene Duplication

“…We identified nine variable AA residues within 10 Å of the center of the binding pocket in the various paralogs (Figure 4, right panel). Site-directed mutagenesis and crystallographic studies by Yamamoto et al confirmed the importance of several of these residues for substrate specificity in the present-day Ima1 protein [39],[40]. In particular, Yamamoto et al [40] characterized the influence of residues 216-217-218 (Ima1 numbering), which covary perfectly with each other and with the observed substrate specificity shifts across the phylogeny presented in Figure 4.…”

Enzymatic assays and molecular modeling studies ofSchisandra chinensislignans and phenolics from fruit and leaf extracts

“…All the x-ray enzyme structures have been downloaded from the Protein Databank RCSB PDB 29 : acetylcholinesterase (pdb:4X3C) 30 in complex with tacrine-nicotinamide hybrid inhibitor, butyrylcholinesterase (pdb:4BDS) 31 in complex with tacrine, amylase (pdb:1VAH) 32 in complex with r-nitrophenyla-D-maltoside, glucosidase (pdb:3AXI) 33 in complex with maltose, and tyrosinase (pdb:2Y9X) 34 in complex with tropolone. The enzymes were separated from their crystallographic inhibitors, also water molecules, ions, and other small molecules were removed by using UCSF Chimera software 35 .…”