Assessment of amino-acid substitutions at tryptophan 16 in alpha-galactosidase

The tryptophan residue at position 16 of coffee bean alpha-galactosidase ha s 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 t ryptophan 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) we re able to support a significant amount of enzyme activity, but the kinetic s and pH profiles of these mutants differed from wild-type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small a mount of enzyme activity with the optimal pH shifted towards more acidic. A ll other residues abolished enzyme activity. Our data support the hypothesi s that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continu ously measuring enzyme kinetics using fluorogenic 4-methylumbelliferyl subs trates. This is useful in screening enzymes from colonies and determining t he enzyme kinetics when the enzyme concentration is not known.