Elastase substrate specificity tailored through substrate-assisted catalysis and phage display.
The catalytic histidine of human neutrophil elastase was replaced with alanine (H57A) to determine if a substrate histidine could substitute for the missing catalytic group-'substrate-assisted catalysis'. H57A and wild-type elastase were recovered directly from Pichia pastoris following expression from a synthetic gene lacking the elastase pro sequence, thereby obviating the need for zymogen activation. Potential histidine-containing substrates for H57A elastase were identified from a phage library of randomized sequences. One such sequence, REHVVY, was cleaved by H57A elastase with a catalytic efficiency, k(cat)/K(M), of 2800 s(-1) M(-1), that is within 160-fold of wild-type elastase. In contrast, wild-type but not H57A elastase cleaved the related non-histidine containing sequence, REAVVY. Ten different histidine-containing linkers were cleaved by H57A elastase. In addition to the requirement for a P2 histidine, significant preferences were observed at other subsites including valine or threonine at P1, and methionine or arginine at P4. A designed sequence, MEHVVY, containing the preferred residues identified at each subsite proved to be a more favorable substrate than any of the phage-derived sequences. Extension of substrate-assisted catalysis to elastase suggests that this engineering strategy may be widely applicable to other serine proteases thereby creating a family of highly specific histidine-dependant proteases.
Dall'Acqua
W
W
Department of Molecular Oncology, Genentech, Inc., 1 DNA Way,South San Francisco, CA 94080, USA.
Halin
C
C
Rodrigues
M L
ML
Carter
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010843:Pichia; D015202:Protein Engineering; D011994:Recombinant Proteins; D013379:Substrate Specificity
D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D019272:Leukocyte Elastase