Human pancreatic α-amylase (HPA) was expressed
in the methylotrophic yeast Pichia pastoris and
two mutants (D197A and D197N) of a completely conserved
active site carboxylic acid were generated. All recombinant
proteins were shown by electrospray ionization mass spectrometry
(ESI-MS) to be glycosylated and the site of attachment
was shown to be Asn461 by peptide mapping in conjunction
with ESI-MS. Treatment of these proteins with endoglycosidase
F demonstrated that they contained a single N-linked oligosaccharide
and yielded a protein product with a single N-acetyl glucosamine
(GlcNAc), which could be crystallized. Solution of the
crystal structure to a resolution of 2.0 Å confirmed
the location of the glycosyl group as Asn461 and showed
that the recombinant protein had essentially the same conformation
as the native enzyme. The kinetic parameters of the glycosylated
and deglycosylated wild-type proteins were the same while
the kcat/Km values
for D197A and D197N were 106–107
times lower than the wild-type enzyme. The decreased
kcat/Km
values for the mutants confirm that D197 plays a crucial
role in the hydrolytic activity of HPA, presumably as the
catalytic nucleophile.