5gch
From Proteopedia
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CHEMISTRY OF CAGED ENZYMES /II$. PHOTOACTIVATION OF INHIBITED CHYMOTRYPSIN
Overview
Inhibited chymotrypsin was reactivated through the photolysis of the covalently bound light-reversible cinnamates described in our previous paper [Stoddard, B.L., Bruhnke, J., Porter, N.A., Ringe, D., & Petsko, G. (1990) Biochemistry 29, 4871-4879]. The light-induced deacylation was accomplished both in solution and in protein crystals, with the release of inhibitor from the crystal monitored and confirmed by X-ray diffraction. The product of photolysis has been characterized as a 3-methylcoumarin, leading to a mechanism for light-driven deacylation of an internal lactonization that is dependent on the presence of an internal hydroxyl nucleophile. The acyl enzyme formed from cinnamate A is not suitable for photochemical studies, as the complex has a short half-life in solution and does not have a chromophore that is well separated from protein absorbance. Cinnamate B, with a p-diethylamino substituent, shows an enzyme deacylation rate enhancement of 10(9) for the cis photoisomer relative to the trans starting material. The half-life and deacylation rate of this compound in the E-I complex after photon absorption have been directly measured by subsecond UV absorption studies. X-ray diffraction studies of photoactivation using a flow cell show that the cinnamate B acyl enzyme complex is fully capable of light-induced isomerization and regeneration of native enzyme in the crystalline state. The E-I complex formed upon binding of cinnamate A, however, shows little if any effect from irradiation due to competitive absorbance by the highly concentrated protein at the shorter UV wavelengths. Photolysis of cinnamate B appears to occur on a time scale fast enough for applications in crystallographic studies of enzymatic intermediate-state structures.
About this Structure
5GCH is a Protein complex structure of sequences from Bos taurus. Active as Chymotrypsin, with EC number 3.4.21.1 Full crystallographic information is available from OCA.
Reference
Photolysis and deacylation of inhibited chymotrypsin., Stoddard BL, Bruhnke J, Koenigs P, Porter N, Ringe D, Petsko GA, Biochemistry. 1990 Sep 4;29(35):8042-51. PMID:2261462
Page seeded by OCA on Thu Feb 21 19:15:15 2008
