title: Human enteropeptidase light chain: bioengineering of recombinants and kinetic investigations of structure and function.
abstract: The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK∼ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (kcat /KM = 6.83 × 10⁶ M⁻¹ sec⁻¹) and DDDDR (kcat /KM = 1.89 × 10⁷ M⁻¹ sec⁻¹) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge-charge interactions in the extended substrate binding pocket.
authors: Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee, USA.
authors: Eliot T
authors: ET
authors: Smith
authors: David A
authors: DA
authors: Johnson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D004765:Enteropeptidase