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Significance of hydrophobic S4-P4 interactions in subtilisin 309 from Bacillus lentus.

Abstract：

:The subtilisins have an extended substrate binding cleft comprising at least 8 subsites. Two pockets at the S1 and S4 sites are particularly conspicuous, and the interactions between substrate and these two pockets are very important for the substrate specificity. Phe residues have mutationally been introduced at one of positions 102, 128, 130, and 132 of the subtilisin Savinase from Bacillus lentus to investigate the effects of introducing bulky groups along the rim of the S4 binding pocket. It is shown that the marked P4 preference of wild-type Savinase for aromatic groups is eliminated by the Gly102-->Phe and Ser128-->Phe mutations, indicating that bulky groups at positions 102 and 128 block the S4 binding site. In contrast, the activity toward hydrophilic P4 residues is not nearly as affected by these mutations, suggesting that the binding mode of the P4 side chain is dependent on its properties. Introduction of a bulky -CH2-S-CH2-CH2-pyridyl group at position 128, by mutational incorporation of Cys followed by chemical modification with 2-vinylpyridine, has essentially the same effect. The Ser130-->Phe mutation hardly affects the activity of the enzyme while the Ser-->Phe mutation at position 132 renders the preference for hydrophobic groups in P4 even more pronounced. This mutation furthermore affects the size of the S4 pocket. An analysis of double mutants at positions 132 and 104 suggests that the S4 region is flexible and is adjusted upon binding of substrates.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Bech LM,Sørensen SB,Breddam K

doi

10.1021/bi00062a016

subject

Has Abstract

pub_date

1993-03-23 00:00:00

pages

2845-52

issue

11

eissn

0006-2960

issn

1520-4995

journal_volume

32

pub_type

杂志文章

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