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Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad.

Abstract：

:The study of proteolysis lies at the heart of our understanding of biocatalysis, enzyme evolution, and drug development. To understand the degree of natural variation in protease active sites, we systematically evaluated simple active site features from all serine, cysteine and threonine proteases of independent lineage. This convergent evolutionary analysis revealed several interrelated and previously unrecognized relationships. The reactive rotamer of the nucleophile determines which neighboring amide can be used in the local oxyanion hole. Each rotamer-oxyanion hole combination limits the location of the moiety facilitating proton transfer and, combined together, fixes the stereochemistry of catalysis. All proteases that use an acyl-enzyme mechanism naturally divide into two classes according to which face of the peptide substrate is attacked during catalysis. We show that each class is subject to unique structural constraints that have governed the convergent evolution of enzyme structure. Using this framework, we show that the γ-methyl of Thr causes an intrinsic steric clash that precludes its use as the nucleophile in the traditional catalytic triad. This constraint is released upon autoproteolysis and we propose a molecular basis for the increased enzymatic efficiency introduced by the γ-methyl of Thr. Finally, we identify several classes of natural products whose mode of action is sensitive to the division according to the face of attack identified here. This analysis of protease structure and function unifies 50 y of biocatalysis research, providing a framework for the continued study of enzyme evolution and the development of inhibitors with increased selectivity.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Buller AR,Townsend CA

doi

10.1073/pnas.1221050110

subject

Has Abstract

pub_date

2013-02-19 00:00:00

pages

E653-61

issue

eissn

0027-8424

issn

1091-6490

pii

1221050110

journal_volume

110

pub_type

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Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad.