Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris

Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic ye ast Pichia pastoris and two mutants (D197A and D197N) of a completely conse rved active site carboxylic acid were generated. All recombinant proteins w ere shown by electrospray ionization mass spectrometry (ESI-MS) to be glyco sylated and the site of attachment was shown to be Asn461 by peptide mappin g in conjunction with ESI-MS. Treatment of these proteins with endoglycosid ase F demonstrated that they contained a single N-linked oligosaccharide an d yielded a protein product with a single N-acetyl glucosamine (GlcNAc), wh ich could be crystallized. Solution of the crystal structure to a resolutio n of 2.0 Angstrom confirmed the location of the glycosyl group as Asn461 an d showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and degly cosylated wild-type proteins were the same while the k(cat)/K-m values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/K-m values for the mutants confirm that D197 plays a cruc ial role in the hydrolytic activity of HPA, presumably as the catalytic nuc leophile.