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Formation of a trypsin-borate-4-aminobutanol ternary complex.

Abstract：

:The formation of ternary complexes involving serine proteases, borate, and an alcohol has important implications for understanding the physiological actions of borate and for the development of tight binding inhibitors for this class of enzymes. Recent studies of a related enzyme, gamma-glutamyl transpeptidase, which is subject to inhibition by a labile serine/borate mixture, have demonstrated that construction of a non-labile boronate analogue results in an inhibitor with nearly 10(5)-fold greater potency. To evaluate the generalization of this biochemistry to serine proteases, we have observed the ternary complex formed from 4-aminobutanol, borate, and trypsin. A combination of (11)B and (1)H NMR and spectrophotometric assays using acetylarginine p-nitroanilide (Ac-Arg-pNA) as the chromogenic substrate all indicate a cooperative binding interaction in which the borate is esterified by the oxygen atoms of the 4-aminobutanol and trypsin residue Ser(195). Two downfield-shifted proton resonances at 15.5 and 16.6 ppm are proposed to arise from the labile imidazolium protons on His(57), indicating a salt bridge interaction with the negatively charged borate. A cooperativity parameter alpha of 0.2 is derived from the assays. These results provide the first direct evidence for formation of a ternary complex involving a serine protease, borate, and an alcohol, and suggest that this represents a general approach for the development of tight binding ligands.

journal_name

Biochemistry

journal_title

Biochemistry

authors

London RE,Gabel SA

doi

10.1021/bi025583z

subject

Has Abstract

pub_date

2002-05-14 00:00:00

pages

5963-7

issue

19

eissn

0006-2960

issn

1520-4995

pii

bi025583z

journal_volume

41

pub_type

杂志文章

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