Observation of the light-triggered binding of pyrone to chymotrypsin by Laue x-ray crystallography. Academic Article uri icon

Overview

abstract

  • Crystals of gamma-chymotrypsin inhibited with the photodissociable group trans-p-diethylamino-o-hydroxy-alpha-methylcinnamate were irradiated with a 1-msec flash from a high-energy xenon flashlamp in the presence of the mechanism-based inhibitor 3-benzyl-6-chloro-2-pyrone. The ensuing reaction was monitored by collection of sequential, single-exposure Laue x-ray diffraction patterns. The experiment was also performed in solution to verify the regeneration of catalytic activity and the subsequent inhibition of the enzyme by pyrone after photolysis. The resulting crystallographic structures show the presence of covalently bound cinnamate prior to photolysis, the generation of "free" enzyme after irradiation of the crystal, and the slow formation of a pyrone-inhibited complex several hours after photolysis. The structure of the free enzyme shows a significant proportion of the active sites in the crystal to contain a naturally occurring, noncovalently bound tetrapeptide inhibitor [Dixon, M.M. & Matthews, B.W. (1989) Biochemistry 28, 7033-7038], even after cinnamate acylation and photolysis. Data collected simultaneously with irradiation show the crystal to be slightly disordered during photolysis, leading to streaked x-ray photos. The resulting maps are suggestive of a bicyclic coumarin species produced by photolysis and deacylation; however, the electron density is difficult to model unambiguously by one unique chemical state. Nevertheless, Laue crystallography is shown to be capable of visualizing time-dependent chemical changes in the active site of an enzyme.

publication date

  • July 1, 1991

Research

keywords

  • Chymotrypsin
  • Pyrones

Identity

PubMed Central ID

  • PMC51905

Scopus Document Identifier

  • 0026075606

Digital Object Identifier (DOI)

  • 10.1073/pnas.88.13.5503

PubMed ID

  • 2062832

Additional Document Info

volume

  • 88

issue

  • 13