Subtilisin BPN' Variants: Increased Hydrolytic Activity on Surface-Bound Substrates via Decreased Surface Activity
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- 作者：Philip F. Brode ; III ; Christopher R. Erwin ; Deborah S. Rauch ; Bobby L. Barnett ; James M. Armpriester ; Ellen S. F. Wang ; and Donn N. Rubingh
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：March 12, 1996
- 年：1996
- 卷：35
- 期：10
- 页码：3162 - 3169
- 全文大小：392K
- 年卷期：v.35,no.10(March 12, 1996)
- ISSN：1520-4995
文摘
Site-directed mutagenesis and random mutagenesis wereused to produce variants of subtilisinBPN' (Bacillus amyloliquefaciens) protease with variablesurface adsorption properties. Protease adsorptionand peptide hydrolysis rate were measured for these variants using amodel substrate consisting of apeptide covalently bound to a surface. While most variants adsorbat a level very similar to that of nativeBPN', several variants were identified which adsorb either more orless. For surface-bound substrateswe report a linear dependence between the concentration of adsorbedprotease enzyme and substratehydrolysis, similar to the linear dependence between enzyme solutionconcentration and hydrolysis ofsoluble substrates. On the basis of this knowledge wehypothesized that variants designed to adsorb ata higher level on a surface-bound peptide substrate would hydrolyzethat surface-bound substrate faster.Contrary to our original expectations, the variants that adsorbmore on the covalently bound peptide surfacehydrolyze this substrate slower. In addition, variants of BPN'which adsorb at a lower level than nativeBPN' hydrolyze the surface-bound substrate faster. Enzymeadsorption and the subsequent peptidehydrolysis are altered by substituting amino acids that modify thesurface charge or hydrophobicity of thenative enzyme. This effect is most dramatic when the changes weremade at surface-exposed sites aroundthe binding pocket/active site of the enzyme. One mechanism thatis consistent with the data is based onthe relationship between the level of adsorption and the enzyme'saffinity for the surface. In this mechanismweakly adsorbed enzymes are postulated to move more rapidly from siteto site on the surface, therebyincreasing substrate hydrolysis.