Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach.
A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.
Oliaro-Bosso
Simonetta
S
Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, Università degli Studi di Torino, Via Pietro Giuria 9, 10125 Turin, Italy.
Schulz-Gasch
Tanja
T
Balliano
Gianni
G
Viola
Franca
F
D001665:Binding Sites; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D019751:Intramolecular Transferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D019751:Intramolecular Transferases; C021924:lanosterol synthase