Abstract：
:Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Brode PF 3rd,Erwin CR,Rauch DS,Barnett BL,Armpriester JM,Wang ES,Rubingh DNdoi
10.1021/bi951990hsubject
Has Abstractpub_date
1996-03-12 00:00:00pages
3162-9issue
10eissn
0006-2960issn
1520-4995pii
bi951990hjournal_volume
35pub_type
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