Open AccessAssessment of amino‐acid substitutions at tryptophan 16 in α‐galactosidase
Author(s) -
Maranville Elizabeth,
Zhu Alex
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01149.x
Subject(s) - tryptophan , enzyme , amino acid , biochemistry , chemistry , cysteine , phenylalanine , tyrosine , residue (chemistry) , enzyme kinetics , lysine , methionine , site directed mutagenesis , active site , enzyme assay , aromatic amino acids , stereochemistry , mutant , gene
The tryptophan residue at position 16 of coffee bean α‐galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site‐directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris , a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild‐type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the p K a of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4‐methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known.