1.The reactivity of alpha-chymotrypsin immobilized on radiation-grafted hydrogel surfaces.
Venkataraman S, Horbett TA, Hoffman AS. J Biomed Mater Res. 1977 Jan;11(1):111-23.
The enzymatic activity of alpha-chymotrypsin (CT), immobilized on hydrogel-coated polymer film supports, has been investigated. The support was prepared by radiation-graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAAc) on silicone rubber films. The enzyme was covalently coupled to the carboxylic group of MAAc via the N-hydroxysuccinimide (NHS) ester active intermediate. Increasing MAAc contents of the hydrogel resulted in increased attachment of CT. The integrity of the CT active site after attachment was assessed by an active site titration with diisopropyl fluorophosphate (DFP). As the MAAc content of the hydrogel was increased, an increasing fraction of the attached CT retained its activity to DFP. A greater fraction of CT was active towards DFP when adsorbed than when coupled. The rates of hydrolysis of some synthetic model substrates by the immobilized CT were also measured. The negative charge on the hydrogel had a large effect on the rates of these hydrolyses.
2.Ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5-3-ketosteroid isomerase by steroid binding.
Zhao Q1, Li YK, Mildvan AS, Talalay P. Biochemistry. 1995 May 16;34(19):6562-72.
delta 5-3-Ketosteroid isomerase (EC 188.8.131.52) from Pseudomonas testosteroni catalyzes the highly efficient conversion of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and intramolecular transfer of the 4 beta-proton to the 6 beta-position. Tyr-14 is the critical general acid and Asp-38 is the general base involved in catalysis. The UV absorption bandwidths of Tyr-14 were much narrower than those of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility. Further immobilization of this residue occurs upon steroid binding. Thus, 5 alpha-estrane-3,17-dione, an A-ring saturated steroid, induces significant narrowing of the tyrosine absorption bands (pi-->pi*) of the main peak (279.5 nm) and the shoulder (285.5 nm) of Tyr-14, with no significant changes in lambda max. No effects of steroid binding were found on the absorption bandwidths of Tyr-55, Tyr-88, or the phenylalanine residues.